Compositions of CXCR4 inhibitors and methods of preparation and use

ABSTRACT

The present invention provides compositions and methods of use for treating, preventing, or ameliorating a disease, disorder, or condition associated with a chemokine receptor such as CXCR4.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds that inhibit C-X-C receptortype 4 (CXCR4). The invention also provides pharmaceutically acceptablecompositions comprising compounds of the present invention and methodsof using said compositions in the treatment of various disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication no. 62,726,010, filed Aug. 31, 2018, the entirety of whichis hereby incorporated by reference.

BACKGROUND OF THE INVENTION

C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or clusterof differentiation 184 (CD184), is a seven transmembrane G-proteincoupled receptor (GPCR) belonging to Class I GPCR or rhodopsin-like GPCRfamily. Under normal physiological conditions, CXCR4 carries outmultiple roles and is principally expressed in the hematopoietic andimmune systems. CXCR4 was initially discovered as one of theco-receptors involved in human immunodeficiency virus (HIV) cell entry.Subsequent studies showed that it is expressed in many tissues,including brain, thymus, lymphatic tissues, spleen, stomach, and smallintestine, and also specific cell types such as hematopoietic stem cells(HSC), mature lymphocytes, and fibroblasts. CXCL12, previouslydesignated SDF-la, is the only known ligand for CXCR4. CXCR4 mediatesmigration of stem cells during embryonic development as well as inresponse to injury and inflammation. Multiple roles have beendemonstrated for CXCR4 in human diseases such as cellular proliferativedisorders, Alzheimer's disease, HIV, rheumatoid arthritis, pulmonaryfibrosis, and others. For example, expression of CXCR4 and CXCL12 havebeen noted in several tumor types. CXCL12 is expressed bycancer-associated fibroblast (CAFs) and is often present at high levelsin the tumor microenvironment (TME). In clinical studies of a wide rangeof tumor types, including breast, ovarian, renal, lung, and melanoma,expression of CXCR4/CXCL12 has been associated with a poor prognosis andwith an increased risk of metastasis to lymph nodes, lung, liver, andbrain, which are sites of CXCL12 expression. CXCR4 is frequentlyexpressed on melanoma cells, particularly the CD133+ population that isconsidered to represent melanoma stem cells; in vitro experiments andmurine models have demonstrated that CXCL12 is chemotactic for suchcells.

Furthermore, there is now evidence implicating the CXCL12/CXCR4 axis incontributing to the loss or lack of tumor responsiveness to angiogenesisinhibitors (also referred to as “angiogenic escape”). In animal cancermodels, interference with CXCR4 function has been demonstrated to alterthe TME and sensitize the tumor to immune attack by multiple mechanismssuch as elimination of tumor re-vascularization and increasing the ratioof CD8+ T cells to Treg cells. These effects result in significantlydecreased tumor burden and increased overall survival in xenograft,syngeneic, and transgenic cancer models. See Vanharanta et al. (2013)Nat Med 19: 50-56; Gale and McColl (1999) BioEssays 21: 17-28; Highfillet al. (2014) Sci Transl Med 6: ra67; Facciabene et al. (2011) Nature475: 226-230.

These data underscore the significant, unmet need for CXCR4 inhibitorsto treat the many diseases and conditions mediated by aberrant orundesired expression of the receptor, for example in cellularproliferative disorders.

SUMMARY OF THE INVENTION

It has now been found that disclosed X4P-001 compositions of the presentinvention, and pharmaceutically acceptable compositions thereof, areeffective as CXC receptor type 4 (CXCR4) inhibitors. In one aspect, thepresent invention provides an X4P-001 composition comprising a compoundof formula I:

or a pharmaceutically acceptable salt thereof; and at least one compoundselected from the following:

or a pharmaceutically acceptable salt thereof.

X4P-001 compositions of the present invention, and pharmaceuticallyacceptable compositions thereof, are useful for treating a variety ofdiseases, disorders, or conditions associated with CXCR4, such ashyperproliferative conditions including various cancers. Such diseases,disorders, or conditions include those described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a detailed summary of the manufacturing process forcapsules containing a solid drug product formulation (unit dosage form)of X4P-001.

FIG. 2 shows a scheme of the original synthesis process (Process version2 or Process 2) used to prepare X4P-001 for clinical trials.

FIG. 3 shows a comparison of Process 3 (the current, improved synthesis)and Process 2 (the original process) for the preparation of X4P-001.

FIG. 4 shows a detailed comparison between Process version 2 and 3 forthe consecutive downstream operations in the work-up and isolation ofthe API.

FIG. 5 shows HPLC and MS data for X4P-001, PTL/ST/0511, batch 3-1(prepared using Process 2), 25° C./60% RH, t=3 months. HPLC conditions 1(described in detail below); 1 mg/mL in methanol, injection volume 100μL.

FIG. 6 shows HPLC and MS data for X4P-001, PTL/ST/0511, batch 3-1(prepared using Process 2), 25° C./60% RH t=3 months. HPLC conditions 2(described in detail below); 1 mg/mL in methanol, injection volume 100μL.

FIG. 7 shows HPLC and MS data for X4P-001, PTL/ST/0511, batch 3-1(prepared using Process 2), 25° C./60% RH t=3 months. HPLC conditions 2(described in detail below); sample concentration 10 mg/mL in methanol,100 μL injection.

FIG. 8 shows HPLC and MS data for X4P-001, degradation sample 80° C./80%RH t=1 day. HPLC conditions 2 (described in detail below); sampleconcentration 10 mg/mL in methanol, 100 μL injection volume.

FIG. 9 shows HPLC and MS data for X4P-001, degradation sample 80° C./80%RH t=7 days. HPLC conditions 2 (described in detail below); sampleconcentration 10 mg/mL in methanol, 100 μL injection volume.

FIG. 10 shows HPLC and MS data for X4P-001, PTL/ST/0511, batch 3-1(prepared using Process 2), 25° C./60% RH t=3 months. HPLC conditions 3(described in detail below); sample concentration 10 mg/mL in methanol,100 μL injection volume.

DETAILED DESCRIPTION OF THE INVENTION 1. General Description of CertainAspects of the Invention

In one aspect, the present invention provides compounds and compositionsthereof useful for treatment, prevention, and/or reduction of a risk ofa disease, disorder, or condition in which CXCR4 is implicated in thepathogenesis. In some embodiments, such compounds include those of theformulae described herein, or a pharmaceutically acceptable saltthereof.

In another aspect, the present invention provides compositions,including formulations and unit dosage forms, comprising X4P-001 (i.e.,a compound of formula I, whose structure is shown below) or apharmaceutically acceptable salt thereof, wherein such compositionsexhibit an improved purity profile. In some embodiments, a disclosedX4P-001 composition exhibits reduced levels of known impurities such asthose described herein, and/or reduced levels of unknown impurities, incomparison with similar compositions prepared by conventional means.

In another aspect, the present invention provides an X4P-001 compositioncomprising a compound of formula I:

or a pharmaceutically acceptable salt thereof; and at least one compoundselected from the following:

or a pharmaceutically acceptable salt thereof.

2. Definitions

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry,” 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of each of which are hereby incorporated by reference.

As used herein, the term “X4P-001 composition” or “disclosed X4P-001composition” refers to a composition comprising a compound of formula I(i.e., X4P-001), or a pharmaceutically acceptable salt thereof, incombination with at least one additional compound selected from I-1,I-2, I-3, I-4, I-5, I-6, or I-7. For clarity, a “pharmaceuticalcomposition” of a disclosed X4P-001 composition refers to a compositioncomprising a compound of formula I (i.e., X4P-001), or apharmaceutically acceptable salt thereof, in combination with at leastone additional compound selected from I-1, I-2, I-3, I-4, I-5, I-6, orI-7, together with a pharmaceutically acceptable excipient, e.g.,adjuvant, filler, binder, carrier, or vehicle.

Additional known or unknown impurities may be present in a disclosedX4P-001 composition. As used herein, the term “impurity” includes one ormore degradants which arise during storage of X4P-001 and/or one or moreby-products formed in a chemical reaction used in manufacturing ofX4P-001. In some embodiments, an impurity results from oxidation,light-initiated decomposition, reaction with a residual solvent such aswater or isopropyl acetate, a side reaction that takes place during theprocess used to prepare X4P-001, or a reaction of X4P-001 with anexcipient present in an X4P-001 pharmaceutical composition.

As used herein, the term “inhibitor” is defined as a compound that bindsto and/or inhibits CXCR4 with measurable affinity. In certainembodiments, an inhibitor has an IC50 and/or binding constant of lessthan about 100 less than about 50 less than about 1 less than about 500nM, less than about 100 nM, less than about 10 nM, or less than about 1nM.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal. describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts include salts of a basic group (e.g. an amino group)formed with inorganic acids such as hydrochloric acid, hydrobromic acid,phosphoric acid, sulfuric acid and perchloric acid or with organic acidssuch as acetic acid, oxalic acid, maleic acid, tartaric acid, citricacid, succinic acid, or malonic acid; or by using other methods used inthe art such as ion exchange. Other pharmaceutically acceptable saltsinclude adipate, alginate, ascorbate, aspartate, benzenesulfonate,besylate, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,glycerophosphate, gluconate, hemi sulfate, heptanoate, hexanoate,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, mesylate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ salts. Representative alkalior alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, (C₁₋₆ alkyl)sulfonate and arylsulfonate.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.

As used herein, a “therapeutically effective amount” or “an effectiveamount” means an amount of a substance (e.g., a therapeutic agent,composition, and/or formulation) that elicits a desired biologicalresponse. In some embodiments, a therapeutically effective amount of asubstance is an amount that is sufficient, when administered as part ofa dosing regimen to a subject suffering from or susceptible to adisease, condition, or disorder, to treat, diagnose, prevent, and/ordelay the onset of the disease, condition, or disorder. As will beappreciated by those of ordinary skill in this art, the effective amountof a substance may vary depending on such factors as the desiredbiological endpoint, the substance to be delivered, the target cell ortissue, etc. For example, the effective amount of compound in aformulation to treat a disease, condition, or disorder is the amountthat alleviates, ameliorates, relieves, inhibits, prevents, delays onsetof, reduces severity of and/or reduces incidence of one or more symptomsor features of the disease, condition, or disorder. In some embodiments,a “therapeutically effective amount” is at least a minimal amount of acompound, or composition containing a compound, which is sufficient fortreating one or more symptoms of a disease or disorder.

As used herein, the terms “treatment,” “treat,” and “treating” refer topartially or completely alleviating, inhibiting, delaying onset of,preventing, ameliorating and/or relieving a disease or disorder, or oneor more symptoms of the disease or disorder, as described herein. Insome embodiments, treatment may be administered after one or moresymptoms have developed. In some embodiments, the term “treating”includes preventing or halting the progression of a disease or disorder.In other embodiments, treatment may be administered in the absence ofsymptoms. For example, treatment may be administered to a susceptibleindividual prior to the onset of symptoms (e.g., in light of a historyof symptoms and/or in light of genetic or other susceptibility factors).Treatment may also be continued after symptoms have resolved, forexample to prevent or delay their recurrence. Thus, in some embodiments,the term “treating” includes preventing relapse or recurrence of adisease or disorder.

As used herein, the term “CXCR4-mediated” in reference to a disorder,disease, and/or condition means any disease, disorder, or condition inwhich CXCR4, or a mutant thereof, is known to play a role. Accordingly,another embodiment of the present invention relates to treating orlessening the severity of one or more diseases in which CXCR4, or amutant thereof, is known to play a role. “CXCR4-mediated” also includesdiseases, disorders, and conditions in which the CXCR4/CXCL12 axis isimplicated.

The term “unit dosage form” as used herein refers to a physicallydiscrete unit of therapeutic formulation appropriate for the subject tobe treated. It will be understood, however, that the total daily usageof the X4P-001 compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular subject or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; activity of specific activeagent employed; specific composition employed; age, body weight, generalhealth, sex and diet of the subject; time of administration, and rate ofexcretion of the specific active agent employed; duration of thetreatment; drugs and/or additional therapies used in combination orcoincidental with specific compound(s) employed, and like factors wellknown in the medical arts.

3. Description of Exemplary Compounds

It has now been found that compounds of the present invention, andcompositions thereof, are useful for treating, preventing, and/orreducing a risk of a disease, disorder, or condition associated withCXCR4 or for which CXCR4 is associated with its pathogenesis.

In one aspect, the present invention provides a composition comprisingX4P-001, or a pharmaceutically acceptable salt thereof, in at least98.5% purity. In some embodiments, the X4P-001 in the composition is atleast 98.5% pure and contains less than 1.5% w/w impurities. In someembodiments, the composition comprises X4P-001 or a pharmaceuticallyacceptable salt thereof in at least 98.6%, 98.7%, 98.8%, 98.9%, 99.0%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% purity.

In one aspect, the present invention provides an X4P-001 compositioncomprising a compound of formula I:

or a pharmaceutically acceptable salt thereof; and a detectable amountof at least one of the following compounds:

or a pharmaceutically acceptable salt thereof and wherein the X4P-001composition does not include the following compound in a detectableamount:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises each of I-1, I-2,I-3, I-5, I-6, and I-7; or a pharmaceutically acceptable salt thereof.

In some embodiments, the amount of I-1, or a pharmaceutically acceptablesalt thereof, is less than about 0.5% w/w of the X4P-001 composition.

In some embodiments, the amount of I-2, or a pharmaceutically acceptablesalt thereof, is less than about 0.3% w/w of the X4P-001 composition.

In some embodiments, the amount of I-3, or a pharmaceutically acceptablesalt thereof, is less than about 0.4% w/w of the X4P-001 composition.

In some embodiments, the amount of I-5, or a pharmaceutically acceptablesalt thereof, is less than about 0.4% w/w of the X4P-001 composition.

In some embodiments, the amount of I-6, or a pharmaceutically acceptablesalt thereof, is less than about 0.4% w/w of the X4P-001 composition.

In some embodiments, the amount of I-7, or a pharmaceutically acceptablesalt thereof, is less than about 0.25% w/w of the X4P-001 composition.

In some embodiments, the amount of I-1, or a pharmaceutically acceptablesalt thereof, is from about 0.02 to about 0.5% w/w of the X4P-001composition.

In some embodiments, the amount of I-2, or a pharmaceutically acceptablesalt thereof, is from about 0.01 to about 0.3% w/w of the X4P-001composition.

In some embodiments, the amount of I-3, or a pharmaceutically acceptablesalt thereof, is from about 0.01 to about 0.4% w/w of the X4P-001composition.

In some embodiments, the amount of I-5, or a pharmaceutically acceptablesalt thereof, is from about 0.01 to about 0.4% w/w of the X4P-001composition.

In some embodiments, the amount of I-6, or a pharmaceutically acceptablesalt thereof, is from about 0.01 to about 0.4% w/w of the X4P-001composition.

In some embodiments, the amount of I-7, or a pharmaceutically acceptablesalt thereof, is from about 0.01 to about 0.25% w/w of the X4P-001composition.

In one aspect, the present invention provides a pharmaceuticalcomposition comprising a disclosed X4P-001 composition, and apharmaceutically acceptable adjuvant, carrier, or vehicle.

In some embodiments, the pharmaceutically acceptable adjuvant includesat least one diluent, a disintegrant, a lubricant, and a flow aid.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a disclosed X4P-001 composition, wherein:

(a) the amount of I-1, or a pharmaceutically acceptable salt thereof, isless than about 0.5% w/w of the X4P-001 composition;

(b) the amount of I-2, or a pharmaceutically acceptable salt thereof, isless than about 0.3% w/w of the X4P-001 composition;

(c) the amount of I-3, or a pharmaceutically acceptable salt thereof, isless than about 0.4% w/w of the X4P-001 composition;

(d) the amount of I-5, or a pharmaceutically acceptable salt thereof, isless than about 0.4% w/w of the X4P-001 composition;

(e) wherein the amount of I-6, or a pharmaceutically acceptable saltthereof, is less than about 0.4% w/w of the X4P-001 composition; and

(f) the amount of I-7, or a pharmaceutically acceptable salt thereof, isless than about 0.25% w/w of the X4P-001 composition.

In one aspect, the present invention provides a pharmaceuticalcomposition comprising a disclosed X4P-001 composition, wherein:

(a) the amount of I-1, or a pharmaceutically acceptable salt thereof, isfrom about 0.02 to about 0.5% w/w of the X4P-001 composition;

(b) the amount of I-2, or a pharmaceutically acceptable salt thereof, isfrom about 0.01 to about 0.3% w/w of the X4P-001 composition;

(c) the amount of I-3, or a pharmaceutically acceptable salt thereof, isfrom about 0.01 to about 0.4% w/w of the X4P-001 composition;

(d) the amount of I-5, or a pharmaceutically acceptable salt thereof, isfrom about 0.01 to about 0.4% w/w of the X4P-001 composition;

(e) the amount of I-6, or a pharmaceutically acceptable salt thereof, isfrom about 0.01 to about 0.4% w/w of the X4P-001 composition; and

(f) the amount of I-7, or a pharmaceutically acceptable salt thereof, isfrom about 0.01 to about 0.25% w/w of the X4P-001 composition.

In another aspect, the present invention provides a unit dosage formcomprising a pharmaceutical composition comprising:

(a) a disclosed X4P-001 composition, in about 10-20% by weight of thecomposition;

(b) microcrystalline cellulose in about 70-85% by weight of thecomposition;

(c) croscarmellose sodium in about 5-10% by weight of the composition;

(d) sodium stearyl fumarate in about 0.5-2% by weight of thecomposition; and

(e) colloidal silicon dioxide in about 0.1-1.0% by weight of thecomposition.

In another aspect, the present invention provides a unit dosage formcomprising a pharmaceutical composition comprising:

(a) a disclosed X4P-001 composition, in about 35-75% by weight of thecomposition;

(b) microcrystalline cellulose in about 5-28% by weight of thecomposition;

(c) dibasic calcium phosphate dihydrate in about 7-30% by weight of thecomposition;

(d) croscarmellose sodium in about 2-10% by weight of the composition;

(e) sodium stearyl fumarate in about 0.3-2.5% by weight of thecomposition;

(f) colloidal silicon dioxide in about 0.05-1.2% by weight of thecomposition; and

(g) sodium lauryl sulfate in about 0.2-1.2% by weight of thecomposition.

In one aspect, the present invention provides a method of treating,preventing, or reducing a risk of a disease, disorder, or conditionassociated with CXCR4 in a subject in need thereof, comprisingadministering to the subject an effective amount of a disclosed X4P-001composition.

In some embodiments, the disease, disorder, or condition is a cancerselected from kidney cancer, renal tumor, renal carcinoma, ovariancancer, or melanoma.

In one aspect, the present invention provides an X4P-001 compositioncomprising a compound of formula I:

or a pharmaceutically acceptable salt thereof; and at least one compoundselected from the following:

or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides an X4P-001 compositioncomprising a compound of formula I:

or a pharmaceutically acceptable salt thereof; and at least one compoundselected from the following:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides an X4P-001composition comprising a compound of formula I:

or a pharmaceutically acceptable salt thereof; and a compound of thefollowing structure:

or a pharmaceutically acceptable salt thereof. In some embodiments, thetotal weight of I-6 and any additional impurities that are presentcomprise no more than about 0.8% w/w of the X4P-001 composition.

In some embodiments, the X4P-001 composition comprises I-6, or apharmaceutically acceptable salt thereof, in at least a detectableamount.

In some embodiments, the X4P-001 composition comprises two, three, orfour compounds selected from I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or apharmaceutically acceptable salt thereof. In some embodiments, theX4P-001 composition comprises at least a detectable amount of two,three, or four compounds selected from I-1, I-2, I-3, I-4, I-5, I-6, orI-7; or a pharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises three compoundsselected from I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or apharmaceutically acceptable salt thereof. In some embodiments, theX4P-001 composition comprises at least a detectable amount of threecompounds selected from I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or apharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises each of I-1, I-2,I-3, I-5, I-6, and I-7; or a pharmaceutically acceptable salt thereof.In some embodiments, the X4P-001 composition comprises at least adetectable amount of each of I-1, I-2, I-3, I-5, I-6, and I-7; or apharmaceutically acceptable salt thereof.

In some embodiments, the total amount of I-6 or a pharmaceuticallyacceptable salt thereof represents no more than about 0.2% w/w of theX4P-001 composition relative to the total weight in the X4P-001composition of the compound of formula I and the one or more compoundsselected from I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or apharmaceutically acceptable salt thereof. In some embodiments, theX4P-001 composition comprises at least a detectable amount of I-6 or apharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises no more than0.15% w/w of I-6 relative to the total weight in the X4P-001 compositionof the compound of formula I and the one or more compounds selected fromI-1, I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceutically acceptablesalt thereof. In some embodiments, the X4P-001 composition comprises atleast a detectable amount of I-6 or a pharmaceutically acceptable saltthereof.

In some embodiments, the one or more compounds selected from I-1, I-2,I-3, I-4, I-5, I-6, or I-7; or a pharmaceutically acceptable saltthereof together represent less than about 3.0% w/w of the X4P-001composition relative to the total weight in the X4P-001 composition ofthe compound of formula I and the one or more compounds selected fromI-1, I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceutically acceptablesalt thereof. In some embodiments, the one or more compounds selectedfrom I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceuticallyacceptable salt thereof are present in at least a detectable amount inthe X4P-001 composition. In some embodiments, the X4P-001 compositioncomprises a compound of formula I, or a pharmaceutically acceptable saltthereof, and does not comprise a compound of formula I-4, or apharmaceutically acceptable salt thereof, in a detectable amount.

In some embodiments, the total organic impurities, including the one ormore compounds selected from I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or apharmaceutically acceptable salt thereof, comprise less than about 4.0%w/w of the X4P-001 composition.

In some embodiments, the amount of I-1, or a pharmaceutically acceptablesalt thereof, is less than about 0.5% w/w of the X4P-001 composition.

In some embodiments, the amount of I-2, or a pharmaceutically acceptablesalt thereof, is less than about 0.3% w/w of the X4P-001 composition.

In some embodiments, the amount of I-3, or a pharmaceutically acceptablesalt thereof, is less than about 0.4% w/w of the X4P-001 composition.

In some embodiments, the X4P-001 composition does not include I-4, or apharmaceutically acceptable salt thereof, in a detectable amount.

In some embodiments, the amount of I-5, or a pharmaceutically acceptablesalt thereof, is less than about 0.07% w/w of the X4P-001 composition.

In some embodiments, the amount of I-6, or a pharmaceutically acceptablesalt thereof, is less than about 0.4% w/w of the X4P-001 composition.

In some embodiments, the amount of I-7, or a pharmaceutically acceptablesalt thereof, is less than about 0.25% w/w of the X4P-001 composition.

In some embodiments, at least a detectable amount of I-1, I-2, I-3, I-5,I-6, or I-7; or a pharmaceutically acceptable salt thereof is present inthe X4P-001 composition.

In some embodiments, the amount of I-1, or a pharmaceutically acceptablesalt thereof, is from about 0.02 to about 0.5% w/w of the X4P-001composition.

In some embodiments, the amount of I-2, or a pharmaceutically acceptablesalt thereof, is from about 0.01 to about 0.3% w/w of the X4P-001composition.

In some embodiments, the amount of I-3, or a pharmaceutically acceptablesalt thereof, is from about 0.01 to about 0.4% w/w of the X4P-001composition.

In some embodiments, the amount of I-5, or a pharmaceutically acceptablesalt thereof, is from about 0.01 to about 0.07% w/w of the X4P-001composition.

In some embodiments, the amount of I-6, or a pharmaceutically acceptablesalt thereof, is from about 0.01 to about 0.4% w/w of the X4P-001composition.

In some embodiments, the amount of I-7, or a pharmaceutically acceptablesalt thereof, is from about 0.01 to about 0.25% w/w of the X4P-001composition.

In some embodiments, the X4P-001 composition comprises the compound offormula I or a pharmaceutically acceptable salt thereof in at least99.3% purity by HPLC and comprises less that 0.7% total additionalcompounds selected from I-1, I-2, I-3, I-5, I-6, or I-7; or apharmaceutically acceptable salt thereof, as measured by HPLC. In someembodiments, at least a detectable amount of one or more of theadditional compounds is present.

In one aspect, the present invention provides an X4P-001 compositioncomprising a compound of formula I:

or a pharmaceutically acceptable salt thereof, and one or more compoundsselected from the following:

or a pharmaceutically acceptable salt thereof, in an amount that is notmore than about 0.5% w/w of the X4P-001 composition;

or a pharmaceutically acceptable salt thereof, in an amount that is notmore than about 0.3% w/w of the X4P-001 composition;

or a pharmaceutically acceptable salt thereof, in an amount that is notmore than about 0.4% w/w of the X4P-001 composition;

or a pharmaceutically acceptable salt thereof, in an amount that is notmore than about 0.5% w/w of the X4P-001 composition;

or a pharmaceutically acceptable salt thereof, in an amount that is notmore than about 0.4% w/w of the X4P-001 composition; or

or a pharmaceutically acceptable salt thereof, in an amount that is notmore than about 0.25% w/w of the X4P-001 composition; wherein each % w/wis measured relative to the total weight in the X4P-001 composition ofthe compound of formula I and the one or more compounds selected fromI-1, I-2, I-3, I-5, I-6, or I-7. In some embodiments, the X4P-001composition comprises a compound of formula I, or a pharmaceuticallyacceptable salt thereof, and does not comprise a compound of formulaI-4, or a pharmaceutically acceptable salt thereof, in a detectableamount.

In some embodiments, the chiral purity of the X4P-001 composition is atleast about 97% enantiomeric excess (% ee).

In some embodiments, the X4P-001 composition comprises 7000, 6000, 5000,4500, 4000, 3500, 3000, 2500, 2000, 1750, 1700, 1650, 1600, 1550, 1500,1450, 1400, or 1350 ppm of toluene or less.

In some embodiments, toluene is used as a crystallization solvent forisolation of X4P-001. In certain embodiments, a specification forresidual toluene in X4P-001 freebase is such that the X4P-001composition comprises no more than 4500 ppm. In other embodiments, theX4P-001 composition comprises no more than 4000 ppm, 3500 ppm, 3000 ppm,2500 ppm, 2000 ppm, 1750 ppm, 1700 ppm, 1650 ppm, 1600 ppm, 1550 ppm,1500 ppm, 1450 ppm, 1400 ppm or 1350 ppm of toluene. In someembodiments, a permitted daily exposure (PDE) approach is used. The termpermitted daily exposure (PDE) is defined as a pharmaceuticallyacceptable intake of residual solvents in a drug. See, e.g., Guidancefor Industry: Q3C Impurities: Residual Solvents published by theDepartment of Health and Human Services, Food and Drug Administration(FDA).

In some embodiments, the % purity of the X4P-001 composition as measuredby HPLC decreases by less than 1% when the X4P-001 composition is storedfor three months at 25° C./60% relative humidity.

In some embodiments, the X4P-001 composition further comprises one ormore of the following:

and wherein compound I-8, I-9, I-10 and/or I-11 are present in an amountless than about 25 parts-per-million (ppm) of the X4P-001 composition.

In some embodiments, compound I-8, I-9, I-10 and/or I-11 are present inan amount less than about 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1 ppmof the X4P-001 composition. In some embodiments, compound I-8, I-9, I-10and/or I-11 are each independently present in an amount between about 1ppm and about 25 ppm, or between about 100 parts-per-billion (ppb) and 4ppm. In some embodiments, the X4P-001 composition comprises a compoundof formula I, or a pharmaceutically acceptable salt thereof, and doesnot comprise a compound of formula I-4, or a pharmaceutically acceptablesalt thereof, in a detectable amount.

In one aspect, the present invention provides a pharmaceuticalcomposition comprising a disclosed X4P-001 composition and apharmaceutically acceptable adjuvant, carrier, or vehicle.

In one aspect, the present invention provides a solid unit dosage formformulated for oral administration comprising a disclosed X4P-001composition or pharmaceutical composition.

In some embodiments, the present invention provides a disclosed X4P-001composition in combination with an additional therapeutic agent.

In another aspect, the present invention provides a unit dosage formcomprising a pharmaceutical composition comprising:

-   -   (a) a disclosed X4P-001 composition, in about 10-20% by weight        of the composition;    -   (b) microcrystalline cellulose in about 70-85% by weight of the        composition;    -   (c) croscarmellose sodium in about 5-10% by weight of the        composition;    -   (d) sodium stearyl fumarate in about 0.5-2% by weight of the        composition; and    -   (e) colloidal silicon dioxide in about 0.1-1.0% by weight of the        composition.

In another aspect, the present invention provides a unit dosage formcomprising a pharmaceutical composition comprising:

-   -   (a) a disclosed X4P-001 composition, in about 30-40% by weight        of the composition;    -   (b) microcrystalline cellulose in about 20-25% by weight of the        composition;    -   (c) dibasic calcium phosphate dihydrate in about 30-35% by        weight of the composition;    -   (d) croscarmellose sodium in about 5-10% by weight of the        composition;    -   (e) sodium stearyl fumarate in about 0.5-2% by weight of the        composition;    -   (f) colloidal silicon dioxide in about 0.1-1.0% by weight of the        composition; and    -   (g) sodium lauryl sulfate in about 0.1-1.0% by weight of the        composition.

In another aspect, the present invention provides a unit dosage formcomprising a pharmaceutical composition comprising:

-   -   (a) a disclosed X4P-001 composition, in about 35-75% by weight        of the composition;    -   (b) microcrystalline cellulose in about 5-28% by weight of the        composition;    -   (c) dibasic calcium phosphate dihydrate in about 7-30% by weight        of the composition;    -   (d) croscarmellose sodium in about 2-10% by weight of the        composition;    -   (e) sodium stearyl fumarate in about 0.3-2.5% by weight of the        composition;    -   (f) colloidal silicon dioxide in about 0.05-1.2% by weight of        the composition; and    -   (g) sodium lauryl sulfate in about 0.2-1.2% by weight of the        composition.

In some embodiments, the unit dosage form is in the form of a capsule.

In some embodiments, the capsule comprises about 25 mg, about 100 mg, orabout 200 mg of X4P-001, or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a method of treating,preventing, or reducing a risk of a disease, disorder, or conditionassociated with CXCR4 in a subject in need thereof, comprisingadministering to the subject a disclosed X4P-001 composition.

In some embodiments, the disease, disorder, or condition is cancer.

In some embodiments, the cancer is selected from kidney cancer, renaltumor, renal carcinoma (including clear cell and papillary renalcarcinoma), ovarian cancer, or melanoma.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and oneadditional compound selected from I-1, I-2, I-3, I-4, I-5, I-6, or I-7;or a pharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and twoadditional compounds selected from I-1, I-2, I-3, I-4, I-5, I-6, or I-7;or a pharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and threeadditional compounds selected from I-1, I-2, I-3, I-4, I-5, I-6, or I-7;or a pharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and fouradditional compounds selected from I-1, I-2, I-3, I-4, I-5, I-6, or I-7;or a pharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and does notcomprise a compound of formula I-4, or a pharmaceutically acceptablesalt thereof, in a detectable amount.

In some embodiments, the additional compound or compounds is present inat least a detectable amount.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and each ofI-1, I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceutically acceptablesalt thereof.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and I-1, or apharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and I-2, or apharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and I-3, or apharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and I-4, or apharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and I-5, or apharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and I-6, or apharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and I-7, or apharmaceutically acceptable salt thereof.

In some embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof; and I-1, I-2,I-3, I-4, I-5, I-6, and I-7; or a pharmaceutically acceptable saltthereof.

In some embodiments, a % w/w amount of a compound in a disclosed X4P-001composition is measured by comparing the area percentage in an HPLCchromatogram of the compound vs. any other compounds present in theX4P-001 composition. For example, if measured in this manner, 0.2% w/wof compound I-6 present in an X4P-001 composition comprising I-6 and acompound of formula I means that the composition contains 0.2% peak area% of I-6 and 99.8% peak area % by HPLC of the compound of formula I. Inother embodiments, the % w/w is measured using another means known toone of ordinary skill in the art, such as those described herein.

In one aspect, the present invention provides a method of preparing adisclosed X4P-001 composition, wherein the composition is preparedsubstantially as described in the Examples and Figures herein.

In another aspect, the present invention provides a compound selectedfrom these depicted in Table 1, below.

TABLE 1 Representative Compounds of the Present Invention

I-1

I-2

I-3

I-4

I-5

I-6

I-7

In some embodiments, the present invention provides a compound depictedin Table 1, above, or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a pharmaceuticalcomposition that comprises a compound of formula I, or apharmaceutically acceptable salt thereof, and at least one of thecompounds depicted in Table 1 above or a pharmaceutically acceptablesalt thereof. The composition may comprise 1, 2, 3, 4, 5, 6, or 7 of thecompounds. In some embodiments, the composition does not comprisecompound I-4 in a detectable amount.

Typically, the wt % of each impurity is determined by HPLC, and ismeasured either initially or after storage, and optionally on anon-going basis during the shelf life of the X4P-001 composition. In someembodiments, the level of an impurity is measured after storage of thecomposition under stressed conditions, which are conditions of elevatedtemperature, humidity, or both, used to approximate the effect oflong-term storage under ambient conditions.

In some embodiments, the compound of formula I or pharmaceuticallyacceptable salt thereof is present in the X4P-001 composition in anamount of at least about 96, 97, 97.5, 98, 98.5, 98.7, 98.9, 99.0, 99.1,99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.75, 99.8, 99.85, 99.9, 99.95,99.97, or 99.999 weight percent where the percentages are based on thefree base of the compound and the total weight of the X4P-001composition. In other embodiments, the X4P-001 composition contains nomore than about 2.0 area percent HPLC of total organic impurities or, inother embodiments, no more than about 5.0, 4.0, 2.0, 1.9, 1.8, 1.7, 1.6,1.5, 1.25, 1, 0.75, 0.5, 0.25, 0.2, 0.1, 0.01, 0.005, or 0.001 areapercent HPLC total organic impurities relative to the total area of theHPLC chromatogram.

In other embodiments, the X4P-001 composition contains no more thanabout 5.0, 4.0, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.25, 1, 0.75, 0.5, 0.25,0.2, 0.1, 0.01, 0.005, or 0.001 area percent (measured by HPLC) ofcompounds I-1, I-2, I-3, I-4, I-5, I-6, and I-7 relative to the totalarea of the HPLC chromatogram.

In other embodiments, the X4P-001 composition comprises a compound offormula I, or a pharmaceutically acceptable salt thereof, and one ormore additional compounds selected from I-1, I-2, I-3, I-4, I-5, I-6, orI-7; or a pharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable carrier. In some embodiments, the X4P-001composition contains the compound of formula I or pharmaceuticallyacceptable salt thereof in an amount of about 1 weight percent to about99 weight percent, where the percentages are based on the free base ofsaid compound and on the total weight of the X4P-001 composition. Inother embodiments, the X4P-001 composition contains no more than about2.0 area percent HPLC of total organic impurities or, in otherembodiments, no more than about 5.0, 4.0, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5,1.25, 1.1, 1.05, 1, 0.95, 0.9, 0.8, 0.75, 0.7, 0.6, 0.5, 0.25, 0.2, 0.1,0.01, 0.005, or 0.001 area percent HPLC total organic impuritiesrelative to the total area of the HPLC chromatogram.

In some embodiments, the one or more compounds selected from I-1, I-2,I-3, I-4, I-5, I-6, or I-7; or a pharmaceutically acceptable saltthereof, together or individually comprise about 0.01-0.20 area percentof the HPLC chromatogram relative to the compound of formula I orpharmaceutically acceptable salt thereof. In some embodiments, the oneor more compounds selected from I-1, I-2, I-3, I-4, I-5, I-6, or I-7; ora pharmaceutically acceptable salt thereof, together or individuallycomprise about 0.02-0.18, 0.03-0.16, 0.05-0.15, 0.075-0.13, 0.09-0.1,0.1-0.2, or 0.15-0.2 area percent of the HPLC chromatogram relative tothe compound of formula I or pharmaceutically acceptable salt thereof.In some embodiments, the foregoing area percentages of the HPLCchromatogram are measured relative to the total area of the HPLCchromatogram instead of relative to the peak area of the compound offormula I or pharmaceutically acceptable salt thereof.

In some embodiments, the one or more compounds selected from I-1, I-2,I-3, I-4, I-5, I-6, or I-7; or a pharmaceutically acceptable saltthereof, together or individually comprise less than about 5.0 weightpercent (% w/w), or about 0.01-5.0% w/w relative to the compound offormula I or pharmaceutically acceptable salt thereof. In someembodiments, the one or more compounds selected from I-1, I-2, I-3, I-4,I-5, I-6, or I-7; or pharmaceutically acceptable salt thereof, togetheror individually comprise less than about 3.0% w/w of the X4P-001composition; or comprise about 0.02-4.0, 0.03-3.5, 0.05-3.1, 0.05-2.9,0.05-2.5, 0.05-2.0, 0.05-1.8, 0.05-1.6, 0.05-1.5, 0.05-1.4, 0.05-1.2,0.05-1.1, 0.05-1.0, 0.05-0.9, 0.05-0.8, 0.05-0.7, 0.05-0.6, 0.05-0.5,0.05-0.4, 0.05-0.3, 0.05-0.2, 0.05-0.1, or about 0.1-0.5% w/w of theX4P-001 composition.

In some embodiments, the total organic impurities, including the one ormore compounds selected from I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or apharmaceutically acceptable salt thereof, comprise less than about 0.05%w/w, about 0.1, 0.5, 1.0, 2.0, 3.0, or about 4.0% w/w or less of theX4P-001 composition. In some embodiments, the total organic impuritiescomprise about 0.02-4.0, 0.03-3.5, 0.05-3.1, 0.05-2.9, 0.05-2.5,0.05-2.0, 0.05-1.8, 0.05-1.6, 0.05-1.5, 0.05-1.4, 0.05-1.2, 0.05-1.1,0.05-1.0, 0.05-0.9, 0.05-0.8, 0.05-0.7, 0.05-0.6, 0.05-0.5, 0.05-0.4,0.05-0.3, 0.05-0.2, 0.05-0.1, or about 0.1-0.5% w/w of the X4P-001composition.

In some embodiments, the amount of I-1, or a pharmaceutically acceptablesalt thereof, is less than about 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,0.02%, or 0.01% w/w of the X4P-001 composition.

In some embodiments, the amount of I-2, or a pharmaceutically acceptablesalt thereof, is less than about 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%,0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% w/w of the X4P-001composition.

In some embodiments, the amount of I-3, or a pharmaceutically acceptablesalt thereof, is less than about 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%,0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% w/w of the X4P-001composition.

In some embodiments, the amount of I-4, or a pharmaceutically acceptablesalt thereof, is less than about 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%,0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% w/w of theX4P-001 composition.

In some embodiments, the amount of I-5, or a pharmaceutically acceptablesalt thereof, is less than about 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%,0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% w/w of theX4P-001 composition.

In some embodiments, the amount of I-6, or a pharmaceutically acceptablesalt thereof, is less than about 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%,0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% w/w of theX4P-001 composition.

In some embodiments, the amount of I-7, or a pharmaceutically acceptablesalt thereof, is less than about 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%,0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% w/w of theX4P-001 composition.

In some embodiments, the amount of I-1, or a pharmaceutically acceptablesalt thereof, is from about 0.001 to about 1.1%, about 0.01 to about1.1%, about 0.01 to about 0.8%, 0.01 to about 0.7%, about 0.01 to about0.6%, 0.01 to about 0.5%, 0.01 to about 0.4%, 0.01 to about 0.3%, 0.01to about 0.2%, 0.01 to about 0.1%, 0.01 to about 0.09%, 0.01 to about0.08%, 0.01 to about 0.07%, 0.01 to about 0.06%, 0.01 to about 0.05%,0.01 to about 0.03%, or 0.01 to about 0.02% w/w of the X4P-001composition.

In some embodiments, the amount of I-2, or a pharmaceutically acceptablesalt thereof, is from about 0.001 to about 0.3%, about 0.01 to about0.3%, 0.01 to about 0.2%, 0.01 to about 0.1%, 0.01 to about 0.09%, 0.01to about 0.08%, 0.01 to about 0.07%, 0.01 to about 0.06%, 0.01 to about0.05%, 0.01 to about 0.03%, or 0.01 to about 0.02% w/w of the X4P-001composition.

In some embodiments, the amount of I-3, or a pharmaceutically acceptablesalt thereof, is from about 0.001 to about 0.4%, about 0.01 to about0.4%, about 0.01 to about 0.3%, 0.01 to about 0.2%, 0.01 to about 0.1%,0.01 to about 0.09%, 0.01 to about 0.08%, 0.01 to about 0.07%, 0.01 toabout 0.06%, 0.01 to about 0.05%, 0.01 to about 0.03%, or 0.01 to about0.02% w/w of the X4P-001 composition.

In some embodiments, the amount of I-4, or a pharmaceutically acceptablesalt thereof, is from about 0.001 to about 0.5%, about 0.01 to about0.5%, about 0.01 to about 0.3%, 0.01 to about 0.2%, 0.01 to about 0.1%,0.01 to about 0.09%, 0.01 to about 0.08%, 0.01 to about 0.07%, 0.01 toabout 0.06%, 0.01 to about 0.05%, 0.01 to about 0.03%, or 0.01 to about0.02% w/w of the X4P-001 composition.

In some embodiments, the amount of I-5, or a pharmaceutically acceptablesalt thereof, is from about 0.001 to about 0.5%, about 0.01 to about0.5%, about 0.01 to about 0.4%, about 0.01 to about 0.3%, 0.01 to about0.2%, 0.01 to about 0.1%, 0.01 to about 0.09%, 0.01 to about 0.08%, 0.01to about 0.07%, 0.01 to about 0.06%, 0.01 to about 0.05%, 0.01 to about0.03%, or 0.01 to about 0.02% w/w of the X4P-001 composition.

In some embodiments, the amount of I-6, or a pharmaceutically acceptablesalt thereof, is from about 0.001 to about 0.5%, about 0.01 to about0.5%, about 0.01 to about 0.4%, about 0.01 to about 0.3%, 0.01 to about0.2%, 0.01 to about 0.1%, 0.01 to about 0.09%, 0.01 to about 0.08%, 0.01to about 0.07%, 0.01 to about 0.06%, 0.01 to about 0.05%, 0.01 to about0.03%, or 0.01 to about 0.02% w/w of the X4P-001 composition.

In some embodiments, the amount of I-7, or a pharmaceutically acceptablesalt thereof, is from about 0.001 to about 0.5%, about 0.01 to about0.5%, about 0.01 to about 0.4%, about 0.01 to about 0.3%, 0.01 to about0.2%, 0.01 to about 0.1%, 0.01 to about 0.09%, 0.01 to about 0.08%, 0.01to about 0.07%, 0.01 to about 0.06%, 0.01 to about 0.05%, 0.01 to about0.03%, or 0.01 to about 0.02% w/w of the X4P-001 composition.

In some embodiments, the amount of any additional or unknown impuritiesin the X4P-001 composition is from about 0.01 to about 0.2% w/w of thecomposition.

In some embodiments, the amount of p-hydroxybenzoic acid present in theX4P-001 composition is from about 0.01 to about 0.5% w/w of thecomposition. In some embodiments, the composition is substantially freeof p-hydroxybenzoic acid. In some embodiments, p-hydroxybenzoic acid isnot present in the composition in a detectable amount. In someembodiments, the X4P-001 composition comprises a compound of formula I,or a pharmaceutically acceptable salt thereof, and does not comprise acompound of formula I-4, or a pharmaceutically acceptable salt thereof,in a detectable amount.

In some embodiments, the chiral purity of the X4P-001 composition is atleast about 97% enantiomeric excess (% ee). In some embodiments, thechiral purity of the compound of formula I is at least 97% ee. In someembodiments, the chiral purity of the compound of formula I is at least98% ee. In some embodiments, the chiral purity of the compound offormula I is at least 99% ee. In some embodiments, the chiral purity ofthe compound of formula I is at least 99.1, 99.2, 99.3, 99.4, 99.5,99.6, 99.7, 99.8, or 99.9% ee.

In some embodiments, the present invention provides any compounddescribed above and herein in isolated form. As used herein, the term“isolated” means that a compound is provided in a form that is separatedfrom other components that might be present in that compound's usualenvironment. In certain embodiments, an isolated compound is in solidform. In some embodiments, an isolated compound is at least about 50%pure as determined by a suitable HPLC method. In certain embodiments, anisolated compound is at least about 60%, 70%, 80%, 90%, 95%, 98%, 99%,99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.99%, or 99.999% pure asdetermined by a suitable HPLC method. Percent purity may be measured byweight percent of the desired compound (% w/w), by area % relative tothe total area of the HPLC chromatogram, or by other methods known inthe art.

Methods of preparation and analysis applicable to certain compounds ofthe invention are disclosed in U.S. Pat. No. 7,354,934, WO 00/56729,U.S. Ser. No. 60/232,891, and U.S. Ser. No. 60/234,510, as well as An,H.; Wang, T.; Mohan, V.; Griffey, R. H.; Cook, P. D. Tetrahedron 1998,54, 3999-4012; the entire contents of each of which is herebyincorporated by reference.

Disclosed compounds may be purified by any means known in the art. Suchmeans include, e.g. silica gel column chromatography; medium pressureliquid chromatography (MPLC); high pressure liquid chromatography(HPLC); preparative HPLC (prep-HPLC); flash chromatography (FC); liquidchromatography (LC); supercritical fluid chromatography (SFC); thinlayer chromatography (TLC); preparative TLC (prep-TLC); liquidchromatography-mass spectrometry (LC-MS, LCMS or LC/MS);recrystallization; precipitation; trituration; distillation;derivitization; acid-base extraction; and the like.

The term “purified,” “in purified form,” or “in isolated and purifiedform” for a compound refers to the physical state of said compound afterbeing isolated from a synthetic process (e.g. from a reaction mixture),or natural source or combination thereof. Thus, the term “purified”, “inpurified form” or “in isolated and purified form” for a compound refersto the physical state of said compound (or a tautomer or stereoisomerthereof, or pharmaceutically acceptable salt or solvate of saidcompound, said stereoisomer, or said tautomer) after being obtained froma purification process or processes described herein or well known tothe skilled artisan (e.g., chromatography, recrystallization and thelike), in sufficient purity to be suitable for in vivo or medicinal useand/or characterizable by standard analytical techniques describedherein or well known to the skilled artisan.

The term “detectable amount,” as used herein, refers to a componentpresent in a sample, for example a sample of a disclosed X4P-001composition, that is present at least in an amount that is capable ofbeing detected by analytical means known in the art. For example, insome embodiments, a “detectable amount” is at least an amount detectableby HPLC, LC-MS, mass spectrometry, NMR, or other analytical methodsknown to one of ordinary skill in the art or described herein.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as, for example, bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereomers to the corresponding pure enantiomers.Enantiomers can also be separated by use of chiral HPLC column.

4. Uses, Formulation, and Administration

Pharmaceutically Acceptable Compositions

In one aspect, the invention provides an X4P-001 composition comprisinga disclosed compound, pharmaceutically acceptable salt, orpharmaceutically acceptable derivative; or a disclosed X4P-001composition; and a pharmaceutically acceptable excipient, carrier,adjuvant, or vehicle. The amount of compound in compositions of thisinvention is such that is effective to measurably inhibit CXCR4, or amutant thereof, in a biological sample or in a patient. In certainembodiments, the amount of compound in compositions of this invention issuch that is effective to measurably inhibit CXCR4, or a mutant thereof,in a biological sample or in a patient. In certain embodiments, acomposition of this invention is formulated for administration to apatient in need of such composition. In some embodiments, a compositionof this invention is formulated for oral administration to a patient.

The term “patient,” as used herein, means an animal, preferably amammal, and most preferably a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

A “pharmaceutically acceptable derivative” means any non-toxic salt,ester, salt of an ester or other derivative of a compound of thisinvention that, upon administration to a recipient, is capable ofproviding, either directly or indirectly, a compound of this inventionor an inhibitorily active metabolite or residue thereof.

As used herein, the term “inhibitorily active metabolite or residuethereof” means that a metabolite or residue thereof is also an inhibitorof CXCR4, or a mutant thereof.

Compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

Pharmaceutically acceptable compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, provided pharmaceutically acceptablecompositions may be formulated in a suitable ointment containing theactive component suspended or dissolved in one or more carriers.Carriers for topical administration of compounds of this inventioninclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, providedpharmaceutically acceptable compositions can be formulated in a suitablelotion or cream containing the active components suspended or dissolvedin one or more pharmaceutically acceptable carriers. Suitable carriersinclude, but are not limited to, mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water.

For ophthalmic use, provided pharmaceutically acceptable compositionsmay be formulated as micronized suspensions in isotonic, pH adjustedsterile saline, or, preferably, as solutions in isotonic, pH adjustedsterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively, for ophthalmic uses, thepharmaceutically acceptable compositions may be formulated in anointment such as petrolatum.

Pharmaceutically acceptable compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Most preferably, pharmaceutically acceptable compositions of thisinvention are formulated for oral administration. Such formulations maybe administered with or without food. In some embodiments,pharmaceutically acceptable compositions of this invention areadministered without food. In other embodiments, pharmaceuticallyacceptable compositions of this invention are administered with food.

The amount of compounds of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the host treated, the particular mode ofadministration. Preferably, provided compositions should be formulatedso that a dosage of between 0.01-100 mg/kg body weight/day of theinhibitor can be administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity of acancer, an autoimmune disorder, a primary immune deficiency, aproliferative disorder, an inflammatory disorder, a neurodegenerative orneurological disorder, schizophrenia, a bone-related disorder, liverdisease, or a cardiac disorder. The exact amount required will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the infection, the particular agent, itsmode of administration, and the like. In some embodiments, compounds ofthe invention are formulated in dosage unit form for ease ofadministration and uniformity of dosage.

The term “patient” or “subject” as used herein, means an animal,preferably a mammal, and most preferably a human.

Pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg or, e.g., from about 1 mg/kg to about 25 mg/kg, of subject bodyweight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

In certain embodiments, the composition is formulated for oraladministration in the form of a tablet or a capsule. In someembodiments, the composition comprising X4P-001 is formulated for oraladministration in the form of a capsule.

In certain embodiments, a provided method comprises administering to thepatient one or more capsules comprising 10 mg to 1200 mg X4P-001 activeingredient; and one or more pharmaceutically acceptable excipients. Incertain embodiments, the capsule is comprised of hard gelatin.

In certain embodiments, the present invention provides a pharmaceuticalcomposition comprising an X4P-001 composition, one or more diluents, adisintegrant, a lubricant, a flow aid, and a wetting agent. In someembodiments, the present invention provides a pharmaceutical compositioncomprising 10 mg to 1200 mg of an X4P-001 composition, microcrystallinecellulose, dibasic calcium phosphate dihydrate, croscarmellose sodium,sodium stearyl fumarate, colloidal silicon dioxide, and sodium laurylsulfate. In some embodiments, the present invention provides a unitdosage form wherein said unit dosage form comprises a pharmaceuticalcomposition comprising 10-200 mg of an X4P-001 composition,microcrystalline cellulose, dibasic calcium phosphate dihydrate,croscarmellose sodium, sodium stearyl fumarate, colloidal silicondioxide, and sodium lauryl sulfate. In certain embodiments, the presentinvention provides a unit dosage form comprising a pharmaceuticalcomposition comprising an X4P-001 composition, present in an amount ofabout 10 mg, about 20 mg, about 25 mg, about 50 mg, about 75 mg, about100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about750 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, orabout 1200 mg. In some embodiments, a provided composition (or unitdosage form) is administered to the patient once per day, twice per day,three times per day, or four times per day. In some embodiments, aprovided composition (or unit dosage form) is administered to thepatient once per day or twice per day.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising:

-   -   (a) a disclosed X4P-001 composition, representing about 30-40%        by weight of the composition;    -   (b) microcrystalline cellulose in about 20-25% by weight of the        composition;    -   (c) dibasic calcium phosphate dihydrate in about 30-35% by        weight of the composition;    -   (d) croscarmellose sodium in about 5-10% by weight of the        composition;    -   (e) sodium stearyl fumarate in about 0.5-2% by weight of the        composition;    -   (f) colloidal silicon dioxide in about 0.1-1.0% by weight of the        composition; and    -   (g) sodium lauryl sulfate in about 0.1-1.0% by weight of the        composition.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising:

-   -   (a) a disclosed X4P-001 composition, representing about 8-25% by        weight of the composition;    -   (b) microcrystalline cellulose in about 65-85% by weight of the        composition;    -   (c) croscarmellose sodium in about 2-10% by weight of the        composition;    -   (d) sodium stearyl fumarate in about 0.1-3% by weight of the        composition; and    -   (e) colloidal silicon dioxide in about 0.05-0.7% by weight of        the composition.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising:

-   -   (a) a disclosed X4P-001 composition, representing about 25-45%        by weight of the composition;    -   (b) microcrystalline cellulose in about 10-35% by weight of the        composition;    -   (c) dibasic calcium phosphate dihydrate in about 15-45% by        weight of the composition;    -   (d) croscarmellose sodium in about 2-10% by weight of the        composition;    -   (e) sodium stearyl fumarate in about 0.3-2.5% by weight of the        composition;    -   (f) colloidal silicon dioxide in about 0.05-1.2% by weight of        the composition; and    -   (g) sodium lauryl sulfate in about 0.2-1.2% by weight of the        composition.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising:

-   -   (a) a disclosed X4P-001 composition, representing about 35-75%        by weight of the composition;    -   (b) microcrystalline cellulose in about 5-28% by weight of the        composition;    -   (c) dibasic calcium phosphate dihydrate in about 7-30% by weight        of the composition;    -   (d) croscarmellose sodium in about 2-10% by weight of the        composition;    -   (e) sodium stearyl fumarate in about 0.3-2.5% by weight of the        composition;    -   (f) colloidal silicon dioxide in about 0.05-1.2% by weight of        the composition; and    -   (g) sodium lauryl sulfate in about 0.2-1.2% by weight of the        composition.

In some embodiments, the X4P-001 composition is present in an amount ofabout 10 mg, about 20 mg, about 25 mg, about 50 mg, about 75 mg, about100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about750 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, orabout 1200 mg.

In some embodiments, the composition comprises about 37 wt % of adisclosed X4P-001 composition.

In some embodiments, the present invention provides a unit dosage formcomprising a disclosed X4P-001 composition or pharmaceuticalcomposition.

In some embodiments, the present invention provides a unit dosage formcomprising a pharmaceutical composition comprising:

-   -   (a) a disclosed X4P-001 composition, in about 10-30% by weight        of the composition;    -   (b) microcrystalline cellulose in about 60-80% by weight of the        composition;    -   (c) croscarmellose sodium in about 5-10% by weight of the        composition;    -   (d) sodium stearyl fumarate in about 0.5-2% by weight of the        composition; and    -   (e) colloidal silicon dioxide in about 0.1-1.0% by weight of the        composition.

In some embodiments, the present invention provides a unit dosage formcomprising a pharmaceutical composition comprising:

-   -   (a) a disclosed X4P-001 composition, in about 14.7% by weight of        the composition;    -   (b) microcrystalline cellulose in about 78.1% by weight of the        composition;    -   (c) croscarmellose sodium in about 6.0% by weight of the        composition;    -   (d) sodium stearyl fumarate in about 1.0% by weight of the        composition; and    -   (e) colloidal silicon dioxide in about 0.2% by weight of the        composition.

In some embodiments, the present invention provides a unit dosage formcomprising a pharmaceutical composition comprising:

-   -   (a) a disclosed X4P-001 composition in about 10-20% by weight of        the composition;    -   (b) microcrystalline cellulose in about 25-40% by weight of the        composition;    -   (c) dibasic calcium phosphate dihydrate in about 35-55% by        weight of the composition;    -   (d) croscarmellose sodium in about 4-15% by weight of the        composition;    -   (e) sodium stearyl fumarate in about 0.3-2% by weight of the        composition;    -   (f) colloidal silicon dioxide in about 0.1-1.5% by weight of the        composition; and    -   (g) sodium lauryl sulfate in about 0.1-1.5% by weight of the        composition.

In some embodiments, the present invention provides a unit dosage formcomprising a pharmaceutical composition comprising:

-   -   (a) a disclosed X4P-001 composition, in about 12.85% by weight        of the composition;    -   (b) microcrystalline cellulose in about 31.92% by weight of the        composition;    -   (c) dibasic calcium phosphate dihydrate in about 44.4% by weight        of the composition;    -   (d) croscarmellose sodium in about 8.33% by weight of the        composition;    -   (e) sodium stearyl fumarate in about 1.38% by weight of the        composition;    -   (f) colloidal silicon dioxide in about 0.42% by weight of the        composition; and    -   (g) sodium lauryl sulfate in about 0.7% by weight of the        composition.

International Patent Application No. PCT/US2016/066634 describesadditional compositions and methods of use of X4P-001, and isincorporated by reference in its entirety.

Inasmuch as it may be desirable to administer a combination of activecompounds, for example, for the purpose of treating a particular diseaseor condition, it is within the scope of the present invention that twoor more pharmaceutical compositions, at least one of which contains acomposition in accordance with the invention, may conveniently becombined in the form of a kit suitable for co-administration of thecompositions. Thus the kit of the invention includes two or moreseparate pharmaceutical compositions, at least one of which contains anX4P-001 composition of the invention, and means for separately retainingsaid compositions, such as a container, divided bottle, or divided foilpacket. An example of such a kit is the familiar blister pack used forthe packaging of tablets, capsules and the like.

The kit of the invention is particularly suitable for administeringdifferent dosage forms, for example, oral and parenteral, foradministering the separate compositions at different dosage intervals,or for titrating the separate compositions against one another. Toassist compliance, the kit typically includes directions foradministration and may be provided with a memory aid.

Uses of Compounds and Pharmaceutically Acceptable Compositions

It has been discovered that certain impurities arise during thesynthesis of X4P-001 such as the compounds shown in Table 1, above, or astereoisomer or pharmaceutically acceptable salt thereof. Isolation andcharacterization of each impurity is useful for a number of purposes.Generally, pharmaceutical compositions require a high level of purity tomeet regulated standards for drug quality and purity. For example, inthe synthesis of X4P-001, impurities are often formed, includingdegradants or by-products of manufacture, which may hinder thetherapeutic effects of X4P-001 and/or may be toxic if present in highenough quantities. As such, it is desirable to have the ability todetermine the presence and amounts of such impurities and to monitor thechemical purity, including stereochemical purity, of X4P-001. To dothis, it is important to identify, isolate, and chemically characterizeimpurities, which can be used in chromatographic procedures as standardsto confirm the purity of X4P-001.

Accordingly, in one aspect, the present invention provides a method ofpreparing a disclosed compound, or a pharmaceutically acceptable saltthereof, comprising contacting an appropriate starting material ormaterials under conditions shown, e.g., in the Examples below, toprepare the compound or pharmaceutically acceptable salt thereof. Insome embodiments, the compound or pharmaceutically acceptable saltthereof is useful as a reference standard and/or in methods ofdetermining the presence of an impurity in a sample, such as a sample ofa compound of formula I, or a pharmaceutically acceptable salt thereof.

The present invention also provides methods for determining an impurity,comprising injecting a reference solution comprising a compound offormula I, or a pharmaceutically acceptable salt thereof, into an HPLCcolumn under a set of conditions to obtain a first HPLC chromatogram,wherein the amount and/or chemical identity of the compound present inthe reference solution is known; injecting a sample solution comprisinga compound of formula I, or a pharmaceutically acceptable salt thereof,into the HPLC column under said set of conditions to obtain a secondHPLC chromatogram; and determining the presence and/or the amount of thecompound in the sample solution. In some embodiments, the referencesolution is injected multiple times. In some embodiments, thedetermining comprises comparing retention times of peaks in the firstHPLC chromatogram and peaks in the second HPLC chromatogram to determinethe presence of the compound in the sample solution. In otherembodiments, the determining comprises quantifying peak areas of thesample solution and peak areas of the reference solution on the HPLCchromatograms and estimating from these the amount of the compound inthe sample solution. In some embodiments, the HPLC column is a reversephase column and the column is eluted using a mobile phase comprisingwater, methanol, trifluoroacetic acid, or mixtures thereof.

The present invention also provides methods for determining the presenceor amount of an impurity in a sample comprising or consistingessentially of a compound of formula I, or a pharmaceutically acceptablesalt thereof, comprising injecting into an HPLC column, in a single orseries of injections, a sample solution containing the material andspiked with a reference compound having a known chemical structure suchas compound I-1, I-2, I-3, I-4, I-5, I-6, or I-7; obtaining an HPLCchromatogram; and determining the presence and/or the amount of thecompound in the material. In some embodiments, the HPLC column is areverse phase column and the column is eluted using a mobile phasecomprising water, methanol, trifluoroacetic acid, or mixtures thereof.The method may further comprise documenting in a written form thechemical identity of the compound and the amount of the compound as animpurity.

In some embodiments, the method further comprises documenting in awritten form the chemical identity of the compound and the amount of thecompound as an impurity in the material. In some cases, the amount inthe material of the compound is determined by (i) identifying a peak onthe chromatogram that corresponds to a peak on a control chromatogram ofa compound known to have the structure of I-1, I-2, I-3, I-4, I-5, I-6,or I-7; (ii) identifying a peak on the chromatogram that corresponds toa relative retention time of a compound known to have the structure ofI-1, I-2, I-3, I-4, I-5, I-6, or I-7; and/or (iii) identifying a peak onthe chromatogram that corresponds to a known amount of a spike of thecompound known to have the structure of I-1, I-2, I-3, I-4, I-5, I-6, orI-7. In some embodiments, the HPLC column is a reverse phase column andthe column is eluted using a mobile phase comprising water, methanol,trifluoroacetic acid, or mixtures thereof.

In some embodiments, the present invention provides a compound selectedfrom I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceuticallyacceptable salt thereof, in sufficient purity in order to enable its useas a reference or standard in various analytical methods (e.g., HPLC,GC, SFC, LCMS), as described more fully below. In some embodiments, thecompound or pharmaceutically acceptable salt thereof may be isolatedwith at least 0.5% purity, at least 1% purity, at least 5% purity, atleast 10% purity, at least 15% purity, at least 25% purity, at least 50%purity, at least 75% purity, at least 95% purity, or with at least 97%purity. In some embodiments, the compound or pharmaceutically acceptablesalt thereof is isolated and/or packaged as a solid.

In another aspect, the present invention provides methods fordetermining the presence and/or amount of I-1, I-2, I-3, I-4, I-5, I-6,or I-7; or a pharmaceutically acceptable salt thereof. For example, thecompound or a pharmaceutically acceptable salt thereof may be formed asan impurity during the synthesis of X4P-001. As used herein, the term“impurity” may refer to degradants which arise during storage of X4P-001and/or by-products formed in a chemical reaction for manufacturing ofX4P-001. In one embodiment, the method comprises injecting a referencesolution comprising I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or apharmaceutically acceptable salt thereof, into an HPLC column under aset of conditions to obtain a first HPLC chromatogram wherein the amountand/or chemical identity of I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or apharmaceutically acceptable salt thereof, present in the referencesolution is known, injecting a sample solution comprising X4P-001 intothe HPLC column under the same set of conditions to obtain a second HPLCchromatogram, and comparing the first HPLC chromatogram with the secondHPLC chromatogram to determine the presence and/or amount of theimpurity (I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceuticallyacceptable salt thereof). The reference solution may be formed bydissolving a sample (e.g., solid sample) of I-1, I-2, I-3, I-4, I-5,I-6, or I-7; or a pharmaceutically acceptable salt thereof, in a firstsolvent, and the sample solution may be formed by dissolving a solidsample in a second solvent. In some embodiments, the reference solutionmay contain an additional compound(s), wherein the amount and/oridentity of the additional compound(s) is also known. In one embodiment,the sample (e.g., sample solution) may comprise X4P-001. It should beunderstood that the invention may encompass other samples suspected ofcontaining a compound selected from I-1, I-2, I-3, I-4, I-5, I-6, orI-7; or a pharmaceutically acceptable salt thereof.

In one embodiment, the presence of I-1, I-2, I-3, I-4, I-5, I-6, or I-7;or a pharmaceutically acceptable salt thereof, in the sample solutionmay be determined by comparing retention times of peaks in the firstHPLC chromatogram with the retention times of peaks in the second HPLCchromatogram. For example, the standard solution comprising I-1, I-2,I-3, I-4, I-5, I-6, or I-7; or a pharmaceutically acceptable saltthereof, may produce a chromatogram with a peak corresponding to I-1,I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceutically acceptable saltthereof, and having a particular retention time. A sample solution maythen be injected into the HPLC column under the same conditions as thestandard solution, and the resulting chromatogram may be studied todetermine if a peak exists at the same retention time as the peakcorresponding to I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or apharmaceutically acceptable salt thereof, in the HPLC chromatogram ofthe standard solution. The existence of such a peak can indicate thatI-1, I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceutically acceptablesalt thereof, is present in the sample. In another embodiment, theamount of I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceuticallyacceptable salt thereof, in the sample solution may be determined bycomparing the area of peaks in the first HPLC chromatogram with the areaof peaks in the second HPLC chromatogram, and calculating from these thecontent of I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceuticallyacceptable salt thereof, in the sample solution.

In some embodiments, the present invention provides methods fordetermining an impurity in a material consisting essentially of X4P-001,wherein a sample solution containing the material and spiked with areference compound having a known chemical structure of I-1, I-2, I-3,I-4, I-5, I-6, or I-7; or a pharmaceutically acceptable salt thereof, asdescribed herein, is injected into an HPLC column and an HPLCchromatogram is obtained to determine the presence and/or the amount ofthe compound in the material.

Methods of the invention may further comprise documenting in a writtenform the chemical identity of the compound and the amount of thecompound as an impurity in the material.

In other embodiments, the present invention provides methods fordetermining an impurity in a material consisting essentially of X4P-001,wherein a solution in which the material is dissolved is injected intoan HPLC column and an HPLC chromatogram is obtained to determine theamount in the material of a compound known to have the structure of I-1,I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceutically acceptable saltthereof, as described herein. The chemical identity of the compound andthe amount of the compound as an impurity in the material may then bedocumented. The amount in the material of the compound may be determinedby (i) identifying a peak on the chromatogram that corresponds to a peakon a control chromatogram, (ii) identifying a peak on the chromatogramthat corresponds to a relative retention time of a compound known tohave the structure of I-1, I-2, I-3, I-4, I-5, I-6, or I-7; orpharmaceutically acceptable salt thereof, and/or (iii) identifying apeak on the chromatogram that corresponds to a known amount of a spikeof the compound known to have the structure of I-1, I-2, I-3, I-4, I-5,I-6, or I-7; or a pharmaceutically acceptable salt thereof.

Some embodiments of the invention may be useful in determining theamount and/or presence of I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or apharmaceutically acceptable salt thereof, in a sample comprisingX4P-001. The sample may be a sample of freshly manufactured material orthe sample may be one stored for a given period of time. In oneembodiment, a sample of X4P-001 may be stored and periodically analyzedusing methods described herein to determine the presence and/or amountof I-1, I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceuticallyacceptable salt thereof, in the sample which may have been formed by,for example, degradation of X4P-001. In some cases, the sample may beplaced under stressed conditions, i.e. conditions to intentionallypromote degradation of X4P-001 such as elevanted temperatures and/orelevated humidity, wherein the sample is periodically analyzed usingmethods described herein to determine the presence and/or amount of I-1,I-2, I-3, I-4, I-5, I-6, or I-7; or a pharmaceutically acceptable saltthereof, in the sample.

Compounds and compositions described herein are generally useful for theinhibition of CXCR4 or a mutant thereof. Certain compounds andcompositions described herein are found to be useful in treatment,prevention, and/or reduction of a risk of a disease, disorder, orcondition associated with CXCR4.

In one aspect, the present invention provides a method of inhibitingCXCR4, or a mutant thereof, activity in a patient comprising the step ofadministering to said patient a disclosed X4P-001 composition. In otherembodiments, the present invention provides a method for treating adisorder mediated by CXCR4, or a mutant thereof, in a patient in needthereof, comprising the step of administering to said patient adisclosed X4P-001 composition according to the present invention or apharmaceutically acceptable composition thereof. Such disorders aredescribed in detail herein.

Certain methods of treating a disease or disorder with X4P-001 aredescribed in PCT Application No. PCT/US2018/038776, filed Jun. 21, 2018,the entirety of which is hereby incorporated by reference. The presentlydisclosed compounds and compositions are applicable in such methods oftreating a disease or disorder.

In certain embodiments, the composition containing X4P-001, or apharmaceutically acceptable salt thereof, is administered orally, in anamount from about 200 mg to about 1200 mg daily. In certain embodiments,the dosage composition may be provided twice a day in divided dosage,approximately 12 hours apart. In other embodiments, the dosagecomposition may be provided once daily. The terminal half-life ofX4P-001 has been generally determined to be between about 12 to about 24hours, or approximately 14.5 hrs. Dosage for oral administration may befrom about 100 mg to about 1200 mg once or twice per day. In certainembodiments, the dosage of X4P-0001, or a pharmaceutically acceptablesalt thereof, useful in the invention is from about 200 mg to about 800mg daily. In other embodiments, the dosage of X4P-001, or apharmaceutically acceptable salt thereof, useful in the invention mayrange from about 200 mg to about 600 mg, from about 400 mg to about 800mg, from about 600 mg to about 1000 mg or from about 800 mg to about1200 mg daily.

In one aspect, the present invention provides a method of treating acancer, such as those described herein, by administering to a patient inneed thereof an effective amount of a disclosed X4P-001 composition. Insome embodiments, the method includes co-administering simultaneously orsequentially an effective amount of one or more additional therapeuticagents, such as those described herein. In some embodiments, the methodincludes co-administering one additional therapeutic agent. In someembodiments, the method includes co-administering two additionaltherapeutic agents. In some embodiments, the combination of thedisclosed compound or composition and the additional therapeutic agentor agents acts synergistically to prevent or reduce immune escape and/orangiogenic escape of the cancer. In some embodiments, the patient haspreviously been administered another anticancer agent, such as anadjuvant therapy or immunotherapy. In some embodiments, the cancer isrefractory.

In some embodiments, the disease, disorder, or condition associated withCXCR4 is selected from cellular proliferative disorders, Alzheimer'sdisease, HIV, rheumatoid arthritis, or pulmonary fibrosis. In someembodiments, the disease, disorder, or condtions is a hyperproliferativedisorder such as cancer. In some embodiments, the cancer is breast,ovarian, renal, lung cancer, or melanoma. In some embodiments, thecancer is selected from renal cell carcinoma (RCC), refractory RCC, orclear cell RCC (ccRCC).

In some embodiments, the present invention provides a method fortreating patients with cancer that presents as a solid tumor. In someembodiments, the patient has kidney cancer, renal tumor, renal carcinoma(including clear cell and papillary renal carcinoma), ovarian cancer, ormelanoma.

Provided compounds are inhibitors of CXCR4 and are therefore useful fortreating one or more disorders associated with activity of CXCR4. Thus,in certain embodiments, the present invention provides a method fortreating a CXCR4-mediated disorder comprising the step of administeringto a patient in need thereof a disclosed X4P-001 composition, orpharmaceutically acceptable composition thereof.

In one aspect, the present invention provides a method of treatingcancer in a patient in need thereof, wherein said method comprisesadministering to said patient a disclosed X4P-001 composition incombination with one or more additional therapeutic agents, such as oneor more immunostimulatory therapeutic compounds.

In some embodiments, the one or more immunostimulatory therapeuticcompounds are selected from elotuzumab, mifamurtide, an agonist oractivator of a toll-like receptor, or an activator of RORyt.

In some embodiments, the method further comprises administering to saidpatient a third therapeutic agent, such as an immune checkpointinhibitor. In some embodiments, the method comprises administering tothe patient in need thereof three therapeutic agents selected from adisclosed X4P-001 composition, an immunostimulatory therapeuticcompound, and an immune checkpoint inhibitor.

In some embodiments, the immune checkpoint inhibitor is selected fromnivolumab, pembrolizumab, ipilimumab, avelumab, durvalumab,atezolizumab, or pidilizumab.

In another aspect, the present invention provides a method of treatingcancer in a patient in need thereof, wherein said method comprisesadministering to said patient a disclosed X4P-001 composition incombination with one or more additional therapeutic agents selected froman indoleamine (2,3)-dioxygenase (IDO) inhibitor, a Poly ADP ribosepolymerase (PARP) inhibitor, a histone deacetylase (HDAC) inhibitor, aCDK4/CDK6 inhibitor, or a phosphatidylinositol 3 kinase (PI3K)inhibitor.

In some embodiments, the IDO inhibitor is selected from epacadostat,indoximod, capmanitib, GDC-0919, PF-06840003, BMS:F001287, Phy906/KD108,or an enzyme that breaks down kynurenine.

In some embodiments, the PARP inhibitor is selected from olaparib,rucaparib, or niraparib.

In some embodiments, the HDAC inhibitor is selected from vorinostat,romidepsin, panobinostat, belinostat, entinostat, or chidamide.

In some embodiments, the CDK 4/6 inhibitor is selected from palbociclib,ribociclib, abemaciclib or trilaciclib.

In some embodiments, the method further comprises administering to saidpatient a third therapeutic agent, such as an immune checkpointinhibitor. In some embodiments, the method comprises administering tothe patient in need thereof three therapeutic agents selected from adisclosed X4P-001 composition, a second therapeutic agent selected froman indoleamine (2,3)-dioxygenase (IDO) inhibitor, a Poly ADP ribosepolymerase (PARP) inhibitor, a histone deacetylase (HDAC) inhibitor, aCDK4/CDK6 inhibitor, or a phosphatidylinositol 3 kinase (PI3K)inhibitor, and a third therapeutic agent selected from an immunecheckpoint inhibitor. In some embodiments, the immune checkpointinhibitor is selected from nivolumab, pembrolizumab, ipilimumab,avelumab, durvalumab, atezolizumab, or pidilizumab.

In some embodiments, the PI3K inhibitor is selected from idelalisib,alpelisib, taselisib, pictilisib, copanlisib, duvelisib, PQR309, orTGR1202.

In another aspect, the present invention provides a method of treatingcancer in a patient in need thereof, wherein said method comprisesadministering to said patient a disclosed X4P-001 composition incombination with one or more additional therapeutic agents selected froma platinum-based therapeutic, a taxane, a nucleoside inhibitor, or atherapeutic agent that interferes with normal DNA synthesis, proteinsynthesis, cell replication, or will otherwise inhibit rapidlyproliferating cells.

In some embodiments, the platinum-based therapeutic is selected fromcisplatin, carboplatin, oxaliplatin, nedaplatin, picoplatin, orsatraplatin.

In some embodiments, the taxane is selected from paclitaxel, docetaxel,albumin-bound paclitaxel, cabazitaxel, or SID530.

In some embodiments, the therapeutic agent that interferes with normalDNA synthesis, protein synthesis, cell replication, or will otherwiseinterfere with the replication of rapidly proliferating cells isselected from trabectedin, mechlorethamine, vincristine, temozolomide,cytarabine, lomustine, azacitidine, omacetaxine mepesuccinate,asparaginase Erwinia chrysanthemi, eribulin mesylate, capacetrine,bendamustine, ixabepilone, nelarabine, clorafabine, trifluridine, ortipiracil.

In some embodiments, the method further comprises administering to saidpatient a third therapeutic agent, such as an immune checkpointinhibitor. In some embodiments, the method comprises administering tothe patient in need thereof three therapeutic agents selected from adisclosed X4P-001 composition, a second therapeutic agent selected froma platinum-based therapeutic, a taxane, a nucleoside inhibitor, or atherapeutic agent that interferes with normal DNA synthesis, proteinsynthesis, cell replication, or will otherwise inhibit rapidlyproliferating cells, and a third therapeutic agent selected from animmune checkpoint inhibitor.

In some embodiments, the immune checkpoint inhibitor is selected fromnivolumab, pembrolizumab, ipilimumab, avelumab, durvalumab,atezolizumab, or pidilizumab.

In some embodiments, any one of the foregoing methods further comprisesthe step of obtaining a biological sample from the patient and measuringthe amount of a disease-related biomarker.

In some embodiments, the biological sample is a blood sample.

In some embodiments, the disease-related biomarker is selected fromcirculating CD8+ T cells or the ratio of CD8+ T cells: Treg cells.

In some embodiments, the cancer is selected from hepatocellularcarcinoma, ovarian cancer, ovarian epithelial cancer, fallopian tubecancer; papillary serous cystadenocarcinoma or uterine papillary serouscarcinoma (UPSC); prostate cancer; testicular cancer; gallbladdercancer; hepatocholangiocarcinoma; soft tissue and bone synovial sarcoma;rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma;anaplastic thyroid cancer; adrenocortical adenoma; pancreatic cancer;pancreatic ductal carcinoma or pancreatic adenocarcinoma;gastrointestinal/stomach (GIST) cancer; lymphoma; squamous cellcarcinoma of the head and neck (SCCHN); salivary gland cancer; glioma,or brain cancer; neurofibromatosis-1 associated malignant peripheralnerve sheath tumors (MPNST); Waldenstrom's macroglobulinemia; ormedulloblastoma.

In some embodiments, the cancer is selected from hepatocellularcarcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovariancancer, ovarian epithelial cancer, fallopian tube cancer, papillaryserous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC),hepatocholangiocarcinoma, soft tissue and bone synovial sarcoma,rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer,adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma,pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associatedmalignant peripheral nerve sheath tumors (MPNST), Waldenstrom'smacroglobulinemia, or medulloblastoma.

In some embodiments, the present invention provides a method fortreating a cancer that presents as a solid tumor, such as a sarcoma,carcinoma, or lymphoma, comprising the step of administering a disclosedX4P-001 composition to a patient in need thereof. Solid tumors generallycomprise an abnormal mass of tissue that typically does not includecysts or liquid areas. In some embodiments, the cancer is selected fromrenal cell carcinoma, or kidney cancer; hepatocellular carcinoma (HCC)or hepatoblastoma, or liver cancer; melanoma; breast cancer; colorectalcarcinoma, or colorectal cancer; colon cancer; rectal cancer; analcancer; lung cancer, such as non-small cell lung cancer (NSCLC) or smallcell lung cancer (SCLC); ovarian cancer, ovarian epithelial cancer,ovarian carcinoma, or fallopian tube cancer; papillary serouscystadenocarcinoma or uterine papillary serous carcinoma (UPSC);prostate cancer; testicular cancer; gallbladder cancer;hepatocholangiocarcinoma; soft tissue and bone synovial sarcoma;rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma;anaplastic thyroid cancer; adrenocortical carcinoma; pancreatic cancer;pancreatic ductal carcinoma or pancreatic adenocarcinoma;gastrointestinal/stomach (GIST) cancer; lymphoma; squamous cellcarcinoma of the head and neck (SCCHN); salivary gland cancer; glioma,or brain cancer; neurofibromatosis-1 associated malignant peripheralnerve sheath tumors (MPNST); Waldenstrom's macroglobulinemia; ormedulloblastoma.

In some embodiments, the cancer is selected from renal cell carcinoma,hepatocellular carcinoma (HCC), hepatoblastoma, colorectal carcinoma,colorectal cancer, colon cancer, rectal cancer, anal cancer, ovariancancer, ovarian epithelial cancer, ovarian carcinoma, fallopian tubecancer, papillary serous cystadenocarcinoma, uterine papillary serouscarcinoma (UPSC), hepatocholangiocarcinoma, soft tissue and bonesynovial sarcoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma,anaplastic thyroid cancer, adrenocortical carcinoma, pancreatic cancer,pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, braincancer, neurofibromatosis-1 associated malignant peripheral nerve sheathtumors (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

In some embodiments, the cancer is selected from hepatocellularcarcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovariancancer, ovarian epithelial cancer, ovarian carcinoma, fallopian tubecancer, papillary serous cystadenocarcinoma, uterine papillary serouscarcinoma (UPSC), hepatocholangiocarcinoma, soft tissue and bonesynovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroidcancer, adrenocortical carcinoma, pancreatic cancer, pancreatic ductalcarcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1associated malignant peripheral nerve sheath tumors (MPNST),Waldenstrom's macroglobulinemia, or medulloblastoma.

In some embodiments, the cancer is hepatocellular carcinoma (HCC). Insome embodiments, the cancer is hepatoblastoma. In some embodiments, thecancer is colon cancer. In some embodiments, the cancer is rectalcancer. In some embodiments, the cancer is ovarian cancer, or ovariancarcinoma. In some embodiments, the cancer is ovarian epithelial cancer.In some embodiments, the cancer is fallopian tube cancer. In someembodiments, the cancer is papillary serous cystadenocarcinoma. In someembodiments, the cancer is uterine papillary serous carcinoma (UPSC). Insome embodiments, the cancer is hepatocholangiocarcinoma. In someembodiments, the cancer is soft tissue and bone synovial sarcoma. Insome embodiments, the cancer is rhabdomyosarcoma. In some embodiments,the cancer is osteosarcoma. In some embodiments, the cancer isanaplastic thyroid cancer. In some embodiments, the cancer isadrenocortical carcinoma. In some embodiments, the cancer is pancreaticcancer, or pancreatic ductal carcinoma. In some embodiments, the canceris pancreatic adenocarcinoma. In some embodiments, the cancer is glioma.In some embodiments, the cancer is malignant peripheral nerve sheathtumors (MPNST). In some embodiments, the cancer is neurofibromatosis-1associated MPNST. In some embodiments, the cancer is Waldenstrom'smacroglobulinemia. In some embodiments, the cancer is medulloblastoma.

In some embodiments, the present invention provides a method fortreating a cancer selected from leukemia or a cancer of the blood,comprising administering to a patient in need thereof an effectiveamount of a disclosed X4P-001 composition, optionally in combinationwith an additional therapeutic agent such as those described herein. Insome embodiments, the cancer is selected from acute myeloid leukemia(AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL),chronic lymphocytic leukemia (CLL), or a virally induced leukemia.

In some embodiments, the patient has a resectable solid tumor, meaningthat the patient's tumor is deemed susceptible to being removed bysurgery. In other embodiments, the patient has an unresectable solidtumor, meaning that the patient's tumor has been deemed not susceptibleto being removed by surgery, in whole or in part.

In some embodiments, the cancer is an advanced cancer, such as anadvanced kidney cancer or advanced renal cell carcinoma.

In some embodiments, the present invention provides a method fortreating refractory cancer in a patient in need thereof comprisingadministering to a patient in need thereof an effective amount of adisclosed X4P-001 composition or pharmaceutical composition thereof,optionally in combination with an additional therapeutic agent such asthose described herein.

In certain embodiments, the patient was previously administered aprotein kinase inhibitor. In some embodiments, the patient waspreviously administered a VEGF-R antagonist. In certain embodiments, thepatient was previously administered an immune checkpoint inhibitor. Insome embodiments, the patient was previously administered an immunecheckpoint inhibitor selected from nivolumab (Opdivo®, Bristol-MyersSquibb), pembrolizumab (Keytruda®, Merck), or ipilumumab (Yervoy®,Bristol-Myers Squibb).

In some embodiments, a disclosed X4P-001 composition is administered toa patient in a fasted state.

Cellular Proliferative Disorders

The present invention features methods and compositions for thediagnosis and prognosis of cellular proliferative disorders (e.g.,cancer) and the treatment of these disorders by targeting CXCR4.Cellular proliferative disorders described herein include, e.g., cancer,obesity, and proliferation-dependent diseases. Such disorders may bediagnosed using methods known in the art.

Cancer

Cancer includes, in one embodiment, without limitation, leukemias (e.g.,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia,acute myeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin'sdisease or non-Hodgkin's disease), Waldenstrom's macroglobulinemia,multiple myeloma, heavy chain disease, and solid tumors such as sarcomasand carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,glioblastoma multiforme (GBM, also known as glioblastoma),medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma,neurofibrosarcoma, meningioma, melanoma, neuroblastoma, andretinoblastoma).

In some embodiments, the cancer is glioma, astrocytoma, glioblastomamultiforme (GBM, also known as glioblastoma), medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma,melanoma, neuroblastoma, or retinoblastoma.

In some embodiments, the cancer is acoustic neuroma, astrocytoma (e.g.Grade I—Pilocytic Astrocytoma, Grade II—Low-grade Astrocytoma, GradeIII—Anaplastic Astrocytoma, or Grade IV—Glioblastoma (GBM)), chordoma,CNS lymphoma, craniopharyngioma, brain stem glioma, ependymoma, mixedglioma, optic nerve glioma, subependymoma, medulloblastoma, meningioma,metastatic brain tumor, oligodendroglioma, pituitary tumors, primitiveneuroectodermal (PNET) tumor, or schwannoma. In some embodiments, thecancer is a type found more commonly in children than adults, such asbrain stem glioma, craniopharyngioma, ependymoma, juvenile pilocyticastrocytoma (JPA), medulloblastoma, optic nerve glioma, pineal tumor,primitive neuroectodermal tumors (PNET), or rhabdoid tumor. In someembodiments, the patient is an adult human. In some embodiments, thepatient is a child or pediatric patient.

Cancer includes, in another embodiment, without limitation,mesothelioma, hepatobilliary (hepatic and billiary duct), bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, ovarian cancer, colon cancer, rectal cancer,cancer of the anal region, stomach cancer, gastrointestinal (gastric,colorectal, and duodenal), uterine cancer, carcinoma of the fallopiantubes, carcinoma of the endometrium, carcinoma of the cervix, carcinomaof the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, testicular cancer,chronic or acute leukemia, chronic myeloid leukemia, lymphocyticlymphomas, cancer of the bladder, cancer of the kidney or ureter, renalcell carcinoma, carcinoma of the renal pelvis, non-Hodgkins's lymphoma,spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocorticalcancer, gall bladder cancer, multiple myeloma, cholangiocarcinoma,fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one ormore of the foregoing cancers.

The present invention further features methods and compositions for thediagnosis, prognosis and treatment of viral-associated cancers,including human immunodeficiency virus (HIV) associated solid tumors,human papilloma virus (HPV)-16 positive incurable solid tumors, andadult T-cell leukemia, which is caused by human T-cell leukemia virustype I (HTLV-I) and is a highly aggressive form of CD4+ T-cell leukemiacharacterized by clonal integration of HTLV-I in leukemic cells (Seehttps://clinicaltrials.gov/ct2/show/study/ NCT02631746); as well asvirus-associated tumors in gastric cancer, nasopharyngeal carcinoma,cervical cancer, vaginal cancer, vulvar cancer, squamous cell carcinomaof the head and neck, and Merkel cell carcinoma. (Seehttps://clinicaltrials.gov/ct2/show/ study/NCT02488759; see alsohttps://clinicaltrials.gov/ct2/show/study/NCT0240886;https://clinicaltrials.gov/ct2/show/NCT02426892)

In some embodiments, the present invention provides a method fortreating a tumor in a patient in need thereof, comprising administeringto the patient a disclosed X4P-001 composition. In some embodiments, thetumor comprises any of the cancers described herein. In someembodiments, the tumor comprises melanoma cancer. In some embodiments,the tumor comprises breast cancer. In some embodiments, the tumorcomprises lung cancer. In some embodiments the the tumor comprises smallcell lung cancer (SCLC). In some embodiments the the tumor comprisesnon-small cell lung cancer (NSCLC).

In some embodiments, the tumor is treated by arresting further growth ofthe tumor. In some embodiments, the tumor is treated by reducing thesize (e.g., volume or mass) of the tumor by at least 5%, 10%, 25%, 50%,75%, 90% or 99% relative to the size of the tumor prior to treatment. Insome embodiments, tumors are treated by reducing the quantity of thetumors in the patient by at least 5%, 10%, 25%, 50%, 75%, 90% or 99%relative to the quantity of tumors prior to treatment.

Primary Immune Deficiencies

In some embodiments, the present invention provides a method fortreating one or more disorders, diseases, and/or conditions wherein thedisorder, disease, or condition includes, but is not limited to, aprimary immunodeficiency disease or disorder, comprising administeringto a patient in need thereof an effective amount of a disclosedcompound. Primary immune deficiencies treatable by the methods of thepresent invention include: warts, hypogammaglobulinemia, infections,myelokathexis (WHIM) syndrome; severe congenital neutropenia (SCN),especially those arising from G6PC3 deficiency (McDermott et al. (2010)Blood 116:2793-2802); GATA2 deficiency (Mono MAC syndrome)(Maciejweski-Duval et al. (2015) J. Leukoc. Biol. 5MA0815-288R (Epub.ahead of printing); idiopathic CD4+T lymphocytopenia (ICL); andWiskott-Aldrich Syndrome.

In other embodiments, the invention relates to a method of inhibitingCXCR4 activity in a biological sample comprising the step of contactingsaid biological sample with a disclosed X4P-001 composition.

According to another embodiment, the invention relates to a method ofinhibiting CXCR4, or a mutant thereof, activity in a biological samplecomprising the step of contacting said biological sample with adisclosed X4P-001 composition. In certain embodiments, the inventionrelates to a method of inhibiting CXCR4, or a mutant thereof, activityin a biological sample comprising the step of contacting said biologicalsample with a disclosed X4P-001 composition.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

Co-Administration with Additional Therapeutic Agents

In one aspect, the present invention provides a method of treating acancer, such as those described herein, by administering to a patient inneed thereof a disclosed X4P-001 composition. In some embodiments, themethod includes co-administering simultaneously or sequentially aneffective amount of one or more additional therapeutic agents, such asthose described herein. In some embodiments, the method includesco-administering one additional therapeutic agent. In some embodiments,the method includes co-administering two additional therapeutic agents.In some embodiments, the combination of a disclosed X4P-001 compositionand the additional therapeutic agent or agents acts synergistically toprevent or reduce immune escape and/or angiogenic escape of the cancer.In some embodiments, the patient has previously been administeredanother anticancer agent, such as an adjuvant therapy or immunotherapy.In some embodiments, the cancer is refractory.

Certain methods of treating a disease or disorder by co-administeringX4P-001 with one or more additional agents are described in PCTApplication No. PCT/US2018/038776, filed Jun. 21, 2018, the entirety ofwhich is hereby incorporated by reference.

Depending upon the particular condition, or disease, to be treated,additional therapeutic agents that are normally administered to treatthat condition, may also be present in the compositions of thisinvention. As used herein, additional therapeutic agents that arenormally administered to treat a particular disease, or condition, areknown as “appropriate for the disease, or condition, being treated.”

In some embodiments, the additional therapeutic agent is a kinaseinhibitor or VEGF-R antagonist. Approved VEGF inhibitors and kinaseinhibitors useful in the present invention include: bevacizumab(Avastin®, Genentech/Roche) an anti-VEGF monoclonal antibody;ramucirumab (Cyramza®, Eli Lilly), an anti-VEGFR-2 antibody andziv-aflibercept, also known as VEGF Trap (Zaltrap®; Regeneron/Sanofi).VEGFR inhibitors, such as regorafenib (Stivarga®, Bayer); vandetanib(Caprelsa®, AstraZeneca); axitinib (Inlyta®, Pfizer); and lenvatinib(Lenvima®, Eisai); Raf inhibitors, such as sorafenib (Nexavar®, Bayer AGand Onyx); dabrafenib (Tafinlar®, Novartis); and vemurafenib (Zelboraf®,Genentech/Roche); MEK inhibitors, such as cobimetanib (Cotellic®,Exelexis/Genentech/Roche); trametinib (Mekinist®, Novartis); Bcr-Abltyrosine kinase inhibitors, such as imatinib (Gleevec®, Novartis);nilotinib (Tasigna®, Novartis); dasatinib (Sprycel®,BristolMyersSquibb); bosutinib (Bosulif®, Pfizer); and ponatinib(Inclusig®, Ariad Pharmaceuticals); Her2 and EGFR inhibitors, such asgefitinib (Iressa®, AstraZeneca); erlotinib (Tarceeva®,Genentech/Roche/Astellas); lapatinib (Tykerb®, Novartis); afatinib(Gilotrif®, Boehringer Ingelheim); osimertinib (targeting activatedEGFR, Tagrisso®, AstraZeneca); and brigatinib (Alunbrig®, AriadPharmaceuticals); c-Met and VEGFR2 inhibitors, such as cabozanitib(Cometriq®, Exelexis); and multikinase inhibitors, such as sunitinib(Sutent®, Pfizer); pazopanib (Votrient®, Novartis); ALK inhibitors, suchas crizotinib (Xalkori®, Pfizer); ceritinib (Zykadia®, Novartis); andalectinib (Alecenza®, Genentech/Roche); Bruton's tyrosine kinaseinhibitors, such as ibrutinib (Imbruvica®, Pharmacyclics/Janssen); andFlt3 receptor inhibitors, such as midostaurin (Rydapt®, Novartis).

Other kinase inhibitors and VEGF-R antagonists that are in developmentand may be used in the present invention include tivozanib (AveoPharmaecuticals); vatalanib (Bayer/Novartis); lucitanib (ClovisOncology); dovitinib (TKI258, Novartis); Chiauanib (ChipscreenBiosciences); CEP-11981 (Cephalon); linifanib (Abbott Laboratories);neratinib (HKI-272, Puma Biotechnology); radotinib (Supect®, IY5511,Il-Yang Pharmaceuticals, S. Korea); ruxolitinib (Jakafi®, IncyteCorporation); PTC299 (PTC Therapeutics); CP-547,632 (Pfizer); foretinib(Exelexis, GlaxoSmithKline); quizartinib (Daiichi Sankyo) and motesanib(Amgen/Takeda).

In some embodiments, the additional therapeutic agent is an mTORinhibitor, which inhibits cell proliferation, angiogenesis and glucoseuptake. Approved mTOR inhibitors useful in the present invention includeeverolimus (Afinitor®, Novartis); temsirolimus (Torisel®, Pfizer); andsirolimus (Rapamune®, Pfizer).

In some embodiments, the additional therapeutic agent is a Poly ADPribose polymerase (PARP) inhibitor. Approved PARP inhibitors useful inthe present invention include olaparib (Lynparza®, AstraZeneca);rucaparib (Rubraca®, Clovis Oncology); and niraparib (Zejula®, Tesaro).Other PARP inhibitors being studied which may be used in the presentinvention include talazoparib (MDV3800/BMN 673/LT00673,Medivation/Pfizer/Biomarin); veliparib (ABT-888, AbbVie); and BGB-290(BeiGene, Inc.).

In some embodiments, the additional therapeutic agent is aphosphatidylinositol 3 kinase (PI3K) inhibitor. Approved PI3K inhibitorsuseful in the present invention include idelalisib (Zydelig®, Gilead).Other PI3K inhibitors being studied which may be used in the presentinvention include alpelisib (BYL719, Novartis); taselisib (GDC-0032,Genentech/Roche); pictilisib (GDC-0941, Genentech/Roche); copanlisib(BAY806946, Bayer); duvelisib (formerly IPI-145, InfinityPharmaceuticals); PQR309 (Piqur Therapeutics, Switzerland); and TGR1202(formerly RP5230, TG Therapeutics).

In some embodiments, the additional therapeutic agent is a proteasomeinhibitor. Approved proteasome inhibitors useful in the presentinvention include bortezomib (Velcade®, Takeda); carfilzomib (Kyprolis®,Amgen); and ixazomib (Ninlaro®, Takeda).

In some embodiments, the additional therapeutic agent is a histonedeacetylase (HDAC) inhibitor. Approved HDAC inhibitors useful in thepresent invention include vorinostat (Zolinza®, Merck); romidepsin(Istodax®, Celgene); panobinostat (Farydak®, Novartis); and belinostat(Beleodaq®, Spectrum Pharmaceuticals). Other HDAC inhibitors beingstudied which may be used in the present invention include entinostat(SNDX-275, Syndax Pharmaceuticals) (NCT00866333); and chidamide(Epidaza®, HBI-8000, Chipscreen Biosciences, China).

In some embodiments, the additional therapeutic agent is a CDKinhibitor, such as a CDK 4/6 inhibitor. Approved CDK 4/6 inhibitorsuseful in the present invention include palbociclib (Ibrance®, Pfizer);and ribociclib (Kisqali®, Novartis). Other CDK 4/6 inhibitors beingstudied which may be used in the present invention include abemaciclib(Ly2835219, Eli Lilly); and trilaciclib (G1T28, G1 Therapeutics).

In some embodiments, the additional therapeutic agent is an indoleamine(2,3)-dioxygenase (IDO) inhibitor. IDO inhibitors being studied whichmay be used in the present invention include epacadostat (INCB024360,Incyte); indoximod (NLG-8189, NewLink Genetics Corporation); capmanitib(INC280, Novartis); GDC-0919 (Genentech/Roche); PF-06840003 (Pfizer);BMS:F001287 (Bristol-Myers Squibb); Phy906/KD108 (Phytoceutica); and anenzyme that breaks down kynurenine (Kynase, Kyn Therapeutics).

In some embodiments, the additional therapeutic agent is a growth factorantagonist, such as an antagonist of platelet-derived growth factor(PDGF), or epidermal growth factor (EGF) or its receptor (EGFR).Approved PDGF antagonists which may be used in the present inventioninclude olaratumab (Lartruvo®; Eli Lilly). Approved EGFR antagonistswhich may be used in the present invention include cetuximab (Erbitux®,Eli Lilly); necitumumab (Portrazza®, Eli Lilly), panitumumab (Vectibix®,Amgen); and osimertinib (targeting activated EGFR, Tagrisso®,AstraZeneca).

In some embodiments, the additional therapeutic agent is an aromataseinhibitor. Approved aromatase inhibitors which may be used in thepresent invention include exemestane (Aromasin®, Pfizer); anastazole(Arimidex®, AstraZeneca) and letrozole (Femara®, Novartis).

In some embodiments, the additional therapeutic agent is an antagonistof the hedgehog pathway. Approved hedgehog pathway inhibitors which maybe used in the present invention include sonidegib (Odomzo®, SunPharmaceuticals); and vismodegib (Erivedge®, Genentech), both fortreatment of basal cell carcinoma.

In some embodiments, the additional therapeutic agent is a folic acidinhibitor. Approved folic acid inhibitors useful in the presentinvention include pemetrexed (Alimta®, Eli Lilly).

In some embodiments, the additional therapeutic agent is a CC chemokinereceptor 4 (CCR4) inhibitor. CCR4 inhibitors being studied that may beuseful in the present invention include mogamulizumab (Poteligeo®, KyowaHakko Kirin, Japan).

In some embodiments, the additional therapeutic agent is an isocitratedehydrogenase (IDH) inhibitor. IDH inhibitors being studied which may beused in the present invention include AG120 (Celgene; NCT02677922);AG221 (Celgene, NCT02677922; NCT02577406); BAY1436032 (Bayer,NCT02746081); IDH305 (Novartis, NCT02987010).

In some embodiments, the additional therapeutic agent is an arginaseinhibitor. Arginase inhibitors being studied which may be used in thepresent invention include AEB1102 (pegylated recombinant arginase,Aeglea Biotherapeutics), which is being studied in Phase 1 clinicaltrials for acute myeloid leukemia and myelodysplastic syndrome(NCT02732184) and solid tumors (NCT02561234); and CB-1158 (CalitheraBiosciences).

In some embodiments, the additional therapeutic agent is a glutaminaseinhibitor. Glutaminase inhibitors being studied which may be used in thepresent invention include CB-839 (Calithera Biosciences).

In some embodiments, the additional therapeutic agent is an antibodythat binds to tumor antigens, that is, proteins expressed on the cellsurface of tumor cells. Approved antibodies that bind to tumor antigenswhich may be used in the present invention include rituximab (Rituxan®,Genentech/BiogenIdec); ofatumumab (anti-CD20, Arzerra®,GlaxoSmithKline); obinutuzumab (anti-CD20, Gazyva®, Genentech),ibritumomab (anti-CD20 and Yttrium-90, Zevalin®, SpectrumPharmaceuticals); daratumumab (anti-CD38, Darzalex®, Janssen Biotech),dinutuximab (anti-glycolipid GD2, Unituxin®, United Therapeutics);trastuzumab (anti-HER2, Herceptin®, Genentech); ado-trastuzumabemtansine (anti-HER2, fused to emtansine, Kadcyla®, Genentech); andpertuzumab (anti-HER2, Perjeta®, Genentech); and brentuximab vedotin(anti-CD30-drug conjugate, Adcetris®, Seattle Genetics).

In some embodiments, the additional therapeutic agent is a topoisomeraseinhibitor. Approved topoisomerase inhibitors useful in the presentinvention include irinotecan (Onivyde®, Merrimack Pharmaceuticals);topotecan (Hycamtin®, GlaxoSmithKline). Topoisomerase inhibitors beingstudied which may be used in the present invention include pixantrone(Pixuvri®, CTI Biopharma).

In some embodiments, the additional therapeutic agent is a nucleosideinhibitor, or other therapeutic that interfere with normal DNAsynthesis, protein synthesis, cell replication, or will otherwiseinhibit rapidly proliferating cells. Such nucleoside inhibitors or othertherapeutics include trabectedin (guanidine alkylating agent, Yondelis®,Janssen Oncology), mechlorethamine (alkylating agent, Valchlor®,Aktelion Pharmaceuticals); vincristine (Oncovin®, Eli Lilly; Vincasar®,Teva Pharmaceuticals; Marqibo®, Talon Therapeutics); temozolomide(prodrug to alkylating agent5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC) Temodar®,Merck); cytarabine injection (ara-C, antimetabolic cytidine analog,Pfizer); lomustine (alkylating agent, CeeNU®, Bristol-Myers Squibb;Gleostine®, NextSource Biotechnology); azacitidine (pyrimidinenucleoside analog of cytidine, Vidaza®, Celgene); omacetaxinemepesuccinate (cephalotaxine ester) (protein synthesis inhibitor,Synribo®; Teva Pharmaceuticals); asparaginase Erwinia chrysanthemi(enzyme for depletion of asparagine, Elspar®, Lundbeck; Erwinaze®, EUSAPharma); eribulin mesylate (microtubule inhibitor, tubulin-basedantimitotic, Halaven®, Eisai); cabazitaxel (microtubule inhibitor,tubulin-based antimitotic, Jevtana®, Sanofi-Aventis); capacetrine(thymidylate synthase inhibitor, Xeloda®, Genentech); bendamustine(bifunctional mechlorethamine derivative, believed to form interstrandDNA cross-links, Treanda®, Cephalon/Teva); ixabepilone (semi-syntheticanalog of epothilone B, microtubule inhibitor, tubulin-basedantimitotic, Ixempra®, Bristol-Myers Squibb); nelarabine (prodrug ofdeoxyguanosine analog, nucleoside metabolic inhibitor, Arranon®,Novartis); clorafabine (prodrug of ribonucleotide reductase inhibitor,competitive inhibitor of deoxycytidine, Clolar®, Sanofi-Aventis); andtrifluridine and tipiracil (thymidine-based nucleoside analog andthymidine phosphorylase inhibitor, Lonsurf®, Taiho Oncology).

In some embodiments, the additional therapeutic agent is aplatinum-based therapeutic, also referred to as platins. Platins causecross-linking of DNA, such that they inhibit DNA repair and/or DNAsynthesis, mostly in rapidly reproducing cells, such as cancer cells.Approved platinum-based therapeutics which may be used in the presentinvention include cisplatin (Platinol®, Bristol-Myers Squibb);carboplatin (Paraplatin®, Bristol-Myers Squibb; also, Teva; Pfizer);oxaliplatin (Eloxitin® Sanofi-Aventis); and nedaplatin (Aqupla®,Shionogi). Other platinum-based therapeutics which have undergoneclinical testing and may be used in the present invention includepicoplatin (Poniard Pharmaceuticals); and satraplatin (JM-216, Agennix).

In some embodiments, the additional therapeutic agent is a taxanecompound, which causes disruption of microtubules, which are essentialfor cell division. Approved taxane compounds which may be used in thepresent invention include paclitaxel (Taxol®, Bristol-Myers Squibb),docetaxel (Taxotere®, Sanofi-Aventis; Docefrez®, Sun Pharmaceutical),albumin-bound paclitaxel (Abraxane®; Abraxis/Celgene), and cabazitaxel(Jevtana®, Sanofi-Aventis). Other taxane compounds which have undergoneclinical testing and may be used in the present invention include SID530(SK Chemicals, Co.) (NCT00931008).

In some embodiments, the additional therapeutic agent is an inhibitor ofanti-apoptotic proteins, such as BCL-2. Approved anti-apoptotics whichmay be used in the present invention include venetoclax (Venclexta®,AbbVie/Genentech); and blinatumomab (Blincyto®, Amgen). Othertherapeutic agents targeting apoptotic proteins which have undergoneclinical testing and may be used in the present invention includenavitoclax (ABT-263, Abbott), a BCL-2 inhibitor (NCT02079740).

The term “checkpoint inhibitor” as used herein relates to agents usefulin preventing cancer cells from avoiding the immune system of thepatient. One of the major mechanisms of anti-tumor immunity subversionis known as “T-cell exhaustion,” which results from chronic exposure toantigens that has led to up-regulation of inhibitory receptors. Theseinhibitory receptors serve as immune checkpoints in order to preventuncontrolled immune reactions.

PD-1 and co-inhibitory receptors such as cytotoxic T-lymphocyte antigen4 (CTLA-4, B and T Lymphocyte Attenuator (BTLA; CD272), T cellImmunoglobulin and Mucin domain-3 (Tim-3), Lymphocyte Activation Gene-3(Lag-3; CD223), and others are often referred to as a checkpointregulators. They act as molecular “gatekeepers” that allow extracellularinformation to dictate whether cell cycle progression and otherintracellular signalling processes should proceed.

In one aspect, the checkpoint inhibitor is a biologic therapeutic or asmall molecule. In another aspect, the checkpoint inhibitor is amonoclonal antibody, a humanized antibody, a fully human antibody, afusion protein or a combination thereof. In a further aspect, thecheckpoint inhibitor inhibits a checkpoint protein selected from CTLA-4,PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR,2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or acombination thereof. In an additional aspect, the checkpoint inhibitorinteracts with a ligand of a checkpoint protein selected from CTLA-4,PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR,2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or acombination thereof. In an aspect, the checkpoint inhibitor is animmunostimulatory agent, a T cell growth factor, an interleukin, anantibody, a vaccine or a combination thereof. In a further aspect, theinterleukin is IL-7 or IL-15. In a specific aspect, the interleukin isglycosylated IL-7. In an additional aspect, the vaccine is a dendriticcell (DC) vaccine.

Checkpoint inhibitors include any agent that blocks or inhibits in astatistically significant manner, the inhibitory pathways of the immunesystem. Such inhibitors may include small molecule inhibitors or mayinclude antibodies, or antigen binding fragments thereof, that bind toand block or inhibit immune checkpoint receptors or antibodies that bindto and block or inhibit immune checkpoint receptor ligands. Illustrativecheckpoint molecules that may be targeted for blocking or inhibitioninclude, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4,BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belongs to the CD2 familyof molecules and is expressed on all NK, γδ, and memory CD8⁺(αβ) Tcells), CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2kinases, A2aR, and various B-7 family ligands. B7 family ligandsinclude, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3,B7-H4, B7-H5, B7-H6 and B7-H7. Checkpoint inhibitors include antibodies,or antigen binding fragments thereof, other binding proteins, biologictherapeutics, or small molecules, that bind to and block or inhibit theactivity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3,GAL9, LAG3, VISTA, KIR, 2B4, CD 160 and CGEN-15049. Illustrative immunecheckpoint inhibitors include Tremelimumab (CTLA-4 blocking antibody),anti-OX40, PD-L1 monoclonal Antibody (Anti-B7-H1; MEDI4736), MK-3475(PD-1 blocker), nivolumab (Opdivo®, BMS-936558; anti-PD1 antibody),CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1antibody), MSB0010718C (anti-PDL1 antibody), and ipilimumab (anti-CTLA-4checkpoint inhibitor). Checkpoint protein ligands include, but are notlimited to PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86 and TIM-3.

In certain embodiments, the immune checkpoint inhibitor is selected froma PD-1 antagonist, a PD-L1 antagonist, or a CTLA-4 antagonist. In someembodiments, a CXCR4 antagonist such as X4P-001 or a pharmaceuticallyacceptable salt thereof is administered in combination with nivolumab(anti-PD-1 antibody, Opdivo®, Bristol-Myers Squibb); pembrolizumab(anti-PD-1 antibody, Keytruda®, Merck); ipilimumab (anti-CTLA-4antibody, Yervoy®, Bristol-Myers Squibb); durvalumab (anti-PD-L1antibody, Imfinzi®, AstraZeneca); or atezolizumab (anti-PD-L1 antibody,Tecentriq®, Genentech).

Other immune checkpoint inhibitors suitable for use in the presentinvention include REGN2810 (Regeneron), an anti-PD-1 antibody tested inpatients with basal cell carcinoma (NCT03132636); NSCLC (NCT03088540);cutaneous squamous cell carcinoma (NCT02760498); lymphoma (NCT02651662);and melanoma (NCT03002376); pidilizumab (CureTech), also known asCT-011, an antibody that binds to PD-1, in clinical trials for diffuselarge B-cell lymphoma and multiple myeloma; avelumab (Bavencio®,Pfizer/Merck KGaA), also known as MSB0010718C), a fully human IgG1anti-PD-L1 antibody, in clinical trials for non-small cell lung cancer,Merkel cell carcinoma, mesothelioma, solid tumors, renal cancer, ovariancancer, bladder cancer, head and neck cancer, and gastric cancer; andPDR001 (Novartis), an inhibitory antibody that binds to PD-1, inclinical trials for non-small cell lung cancer, melanoma, triplenegative breast cancer and advanced or metastatic solid tumors.Tremelimumab (CP-675,206; Astrazeneca) is a fully human monoclonalantibody against CTLA-4 that has been in studied in clinical trials fora number of indications, including: mesothelioma, colorectal cancer,kidney cancer, breast cancer, lung cancer and non-small cell lungcancer, pancreatic ductal adenocarcinoma, pancreatic cancer, germ cellcancer, squamous cell cancer of the head and neck, hepatocellularcarcinoma, prostate cancer, endometrial cancer, metastatic cancer in theliver, liver cancer, large B-cell lymphoma, ovarian cancer, cervicalcancer, metastatic anaplastic thyroid cancer, urothelial cancer,fallopian tube cancer, multiple myeloma, bladder cancer, soft tissuesarcoma, and melanoma. AGEN-1884 (Agenus) is an anti-CTLA4 antibody thatis being studied in Phase 1 clinical trials for advanced solid tumors(NCT02694822).

Nivolumab (Opdivo®, BMS-93568/MDX1106; Bristol-Myers Squibb), is a fullyhuman IgG4 monoclonal antibody that acts as an immunomodulator bybinding to the programmed cell death 1 (PD-1) receptor and selectivelyblocking interaction with its ligands PD-L1 and PD-L2. The structure andother properties of nivolumab are specified athttp://www.drugbank.ca/drugs/DB09035, accessed on Mar. 14, 2016, thedisclosure of which is hereby incorporated herein. Nivolumab is approvedfor use in treatment of patients with advanced renal cell carcinoma whohave received prior anti-angiogenic therapy; as a single agent incertain types of unresectable or metastatic melanoma; in treatingunresectable or metastatic melanoma or in combination with ipilimumab intreating unresectable or metastatic melanoma; and for treatment ofmetastatic non-small cell lung cancer and progression on or afterplatinum-based chemotherapy. Additionally, nivolumab has been tested ormentioned as a possible treatment in other oncologic indications,including solid tumors; skin melanoma; glioblastoma; glioma;gliosarcoma; astrocytoma; brain cancer; leukemia; acute myeloidleukemia; chronic myeloid leukemia; chronic lymphocytic leukemia;advanced liver cancer or hepatocellular carcinoma; uveal melanoma;prostate cancer; pancreatic neoplasm and pancreatic cancer; bladdercancer; colorectal cancer; myelodysplastic syndrome; Hodgkin Lymphoma;Non-Hodgkin Lymphoma; multiple myeloma; cervical cancer; endometrialcancer; uterine cancer; ovarian cancer and ovarian carcinoma; peritonealcarcinoma; head and neck squamous cell cancer; gastric cancer;esophageal cancer; Kaposi sarcoma; breast neoplasm, breastadenocarcinoma and breast cancer; bone sarcoma; soft tissue sarcoma;meningiomas; and mesothelioma.

In a phase 3 trial of over 800 patients with advanced clear-cell renalcell carcinoma, for which they had received previous treatment with oneor two regimens of antiangiogenic therapy, were randomly assigned toreceive 3 mg/kg body weight of nivolumab, intravenously every two weeks,or a 10 mg everolimus tablet orally daily. Patients treated withnivolumab exhibited longer median overall survival, decreased hazardratio for death, and higher objective response rate than those patientstreated with nivolumab (25%) compared to everolimus (5%) (P<0.001), withlower incidence of Grade 3 or 4 treatment-related adverse events (Motzeret al. (2015), New England Journal of Medicine, 373:1803-1813).Accordingly, in some embodiments, the present invention provides amethod of treating advanced clear-cell renal cell carcinoma, comprisingadministering to a patient in need thereof an effective amount of aCXCR4 antagonist such as X4P-001 or a pharmaceutically acceptable saltthereof or pharmaceutical composition thereof in combination withnivolumab or everolimus, optionally wherein that patient has receivedprevious treatment with a regimen of antiangiogenic therapy.

Generally, the amount of nivolumab or other immune checkpoint inhibitoruseful in the present invention will be dependent upon the size, weight,age and condition of the patient being treated, the severity of thedisorder or condition, and the discretion of the prescribing physician.For example, in its current prescribed labeling for unresectable ormetastatic renal cell carcinoma, the recommended course ofadministration for nivolumab is 3 mg/kg as an intravenous infusion over60 minutes every two weeks, until disease progression or unacceptabletoxicity. In the discretion of the clinician, depending upon individualtolerance, the prescribed dose of nivolumab may be increased, forexample, increased in dosage and/or frequency. In the discretion of theclinician, together with the warnings provided with prescribinginformation, administration of nivolumab may be discontinued, or thedose reduced in the case of significant adverse effects. In someembodiments, nivolumab is administered in the methods of the presentinvention according to the labeling guidelines above.

In some embodiments, the present invention provides a method fortreating a patient by administering a CXCR4 antagonist such as X4P-001or a pharmaceutically acceptable salt thereof in combination with animmunostimulatory therapeutics. Approved immunostimulatory therapeuticswhich may be used in the present invention include elotuzumab(anti-SLAMF7-antibody, Empliciti®, Bristol-Myers Squibb).Immunostimulatory compounds being studied that may be used in thepresent invention include mifamurtide (Mepact®, Takeda Oncology).

Another immunostimulatory therapeutic that may be used in the presentinvention is recombinant human interleukin 15 (rhIL-15). rhIL-15 hasbeen tested in the clinic as a therapy for melanoma and renal cellcarcinoma (NCT01021059 and NCT01369888) and leukemias (NCT02689453).Another immunostimulatory therapeutic that may be used in the presentinvention is recombinant human interleukin 12 (rhlL-12). Anothersuitable IL-15 based immunotherapeutic is heterodimeric IL-15 (hetIL-15,Novartis/Admune), a fusion complex composed of a synthetic form ofendogenous IL-15 complexed to the soluble IL-15 binding protein IL-15receptor alpha chain (IL15:sIL-15RA), which has been tested in Phase 1clinical trials for melanoma, renal cell carcinoma, non-small cell lungcancer and head and neck squamous cell carcinoma (NCT02452268).Recombinant human interleukin 12 (rhlL-12) has been tested in the clinicfor many oncological indications, for example, as a therapy for lymphoma(NM-IL-12, Neumedicines, Inc.), (NCT02544724 and NCT02542124).

Another paradigm for immune-stimulation is the use of oncolytic viruses.In some embodiments, the present invention provides a method fortreating a patient by administering a disclosed X4P-001 composition incombination with an immunostimulatory therapy such as oncolytic viruses.Approved immunostimulatory oncolytic viruses which may be used in thepresent invention include talimogene laherparepvec (live, attenuatedherpes simplex virus, Imlygic®, Amgen).

The term “aromatase inhibitor” as used herein relates to a compoundwhich inhibits estrogen production, for instance, the conversion of thesubstrates androstenedione and testosterone to estrone and estradiol,respectively. The term includes, but is not limited to steroids,especially atamestane, exemestane and formestane and, in particular,non-steroids, especially aminoglutethimide, roglethimide,pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole,fadrozole, anastrozole and letrozole. Exemestane is marketed under thetrade name Aromasin™. Formestane is marketed under the trade nameLentaron™. Fadrozole is marketed under the trade name Afema™.Anastrozole is marketed under the trade name Arimidex™. Letrozole ismarketed under the trade names Femara™ or Femar™. Aminoglutethimide ismarketed under the trade name Orimeten™ A combination of the inventioncomprising a chemotherapeutic agent which is an aromatase inhibitor isparticularly useful for the treatment of hormone receptor positivetumors, such as breast tumors.

The term “antiestrogen” as used herein relates to a compound whichantagonizes the effect of estrogens at the estrogen receptor level. Theterm includes, but is not limited to tamoxifen, fulvestrant, raloxifeneand raloxifene hydrochloride. Tamoxifen is marketed under the trade nameNolvadex™. Raloxifene hydrochloride is marketed under the trade nameEvista™. Fulvestrant can be administered under the trade name Faslodex™.A combination of the invention comprising a chemotherapeutic agent whichis an antiestrogen is particularly useful for the treatment of estrogenreceptor positive tumors, such as breast tumors.

The term “anti-androgen” as used herein relates to any substance whichis capable of inhibiting the biological effects of androgenic hormonesand includes, but is not limited to, bicalutamide (Casodex™). The term“gonadorelin agonist” as used herein includes, but is not limited toabarelix, goserelin and goserelin acetate. Goserelin can be administeredunder the trade name Zoladex™

The term “topoisomerase I inhibitor” as used herein includes, but is notlimited to topotecan, gimatecan, irinotecan, camptothecian and itsanalogues, 9-nitrocamptothecin and the macromolecular camptothecinconjugate PNU-166148. Irinotecan can be administered, e.g. in the formas it is marketed, e.g. under the trademark Camptosar™ Topotecan ismarketed under the trade name Hycamptin™.

The term “topoisomerase II inhibitor” as used herein includes, but isnot limited to the anthracyclines such as doxorubicin (includingliposomal formulation, such as Caelyx™), daunorubicin, epirubicin,idarubicin and nemorubicin, the anthraquinones mitoxantrone andlosoxantrone, and the podophillotoxines etoposide and teniposide.Etoposide is marketed under the trade name Etopophos™. Teniposide ismarketed under the trade name VM 26-Bristol Doxorubicin is marketedunder the trade name Acriblastin™ or Adriamycin™. Epirubicin is marketedunder the trade name Farmorubicin™. Idarubicin is marketed. under thetrade name Zavedos™. Mitoxantrone is marketed under the trade nameNovantron.

The term “microtubule active agent” relates to microtubule stabilizing,microtubule destabilizing compounds and microtublin polymerizationinhibitors including, but not limited to taxanes, such as paclitaxel anddocetaxel; vinca alkaloids, such as vinblastine or vinblastine sulfate,vincristine or vincristine sulfate, and vinorelbine; discodermolides;cochicine and epothilones and derivatives thereof. Paclitaxel ismarketed under the trade name Taxol™. Docetaxel is marketed under thetrade name Taxotere™. Vinblastine sulfate is marketed under the tradename Vinblastin R.P™. Vincristine sulfate is marketed under the tradename Farmistin™.

The term “alkylating agent” as used herein includes, but is not limitedto, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNU orGliadel). Cyclophosphamide is marketed under the trade name Cyclostin™.Ifosfamide is marketed under the trade name Holoxan™.

The term “histone deacetylase inhibitors” or “HDAC inhibitors” relatesto compounds which inhibit the histone deacetylase and which possessantiproliferative activity. This includes, but is not limited to,suberoylanilide hydroxamic acid (SAHA).

The term “antineoplastic antimetabolite” includes, but is not limitedto, 5-fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylatingcompounds, such as 5-azacytidine and decitabine, methotrexate andedatrexate, and folic acid antagonists such as pemetrexed. Capecitabineis marketed under the trade name Xeloda™. Gemcitabine is marketed underthe trade name Gemzar™.

The term “platin compound” as used herein includes, but is not limitedto, carboplatin, cis-platin, cisplatinum and oxaliplatin. Carboplatincan be administered, e.g., in the form as it is marketed, e.g. under thetrademark Carboplat™. Oxaliplatin can be administered, e.g., in the formas it is marketed, e.g. under the trademark Eloxatin™.

The term “compounds targeting/decreasing a protein or lipid kinaseactivity; or a protein or lipid phosphatase activity; or furtheranti-angiogenic compounds” as used herein includes, but is not limitedto, protein tyrosine kinase and/or serine and/or threonine kinaseinhibitors or lipid kinase inhibitors, such as a) compounds targeting,decreasing or inhibiting the activity of the platelet-derived growthfactor-receptors (PDGFR), such as compounds which target, decrease orinhibit the activity of PDGFR, especially compounds which inhibit thePDGF receptor, such as an N-phenyl-2-pyrimidine-amine derivative, suchas imatinib, SU101, SU6668 and GFB-111; b) compounds targeting,decreasing or inhibiting the activity of the fibroblast growthfactor-receptors (FGFR); c) compounds targeting, decreasing orinhibiting the activity of the insulin-like growth factor receptor I(IGF-IR), such as compounds which target, decrease or inhibit theactivity of IGF-IR, especially compounds which inhibit the kinaseactivity of IGF-I receptor, or antibodies that target the extracellulardomain of IGF-I receptor or its growth factors; d) compounds targeting,decreasing or inhibiting the activity of the Trk receptor tyrosinekinase family, or ephrin B4 inhibitors; e) compounds targeting,decreasing or inhibiting the activity of the AxI receptor tyrosinekinase family; f) compounds targeting, decreasing or inhibiting theactivity of the Ret receptor tyrosine kinase; g) compounds targeting,decreasing or inhibiting the activity of the Kit/SCFR receptor tyrosinekinase, such as imatinib; h) compounds targeting, decreasing orinhibiting the activity of the C-kit receptor tyrosine kinases, whichare part of the PDGFR family, such as compounds which target, decreaseor inhibit the activity of the c-Kit receptor tyrosine kinase family,especially compounds which inhibit the c-Kit receptor, such as imatinib;i) compounds targeting, decreasing or inhibiting the activity of membersof the c-Abl family, their gene-fusion products (e.g. BCR-Abl kinase)and mutants, such as compounds which target decrease or inhibit theactivity of c-Abl family members and their gene fusion products, such asan N-phenyl-2-pyrimidine-amine derivative, such as imatinib or nilotinib(AMN107); PD180970; AG957; NSC 680410; PD173955 from ParkeDavis; ordasatinib (BMS-354825); j) compounds targeting, decreasing or inhibitingthe activity of members of the protein kinase C (PKC) and Raf family ofserine/threonine kinases, members of the MEK, SRC, JAK/pan-JAK, FAK,PDK1, PKB/Akt, Ras/MAPK, PI3K, SYK, TYK2, BTK and TEC family, and/ormembers of the cyclin-dependent kinase family (CDK) includingstaurosporine derivatives, such as midostaurin; examples of furthercompounds include UCN-01, safingol, BAY 43-9006, Bryostatin 1,Perifosine; llmofosine; RO 318220 and RO 320432; GO 6976; lsis 3521;LY333531/LY379196; isochinoline compounds; FTIs; PD184352 or QAN697 (aP13K inhibitor) or AT7519 (CDK inhibitor); k) compounds targeting,decreasing or inhibiting the activity of protein-tyrosine kinaseinhibitors, such as compounds which target, decrease or inhibit theactivity of protein-tyrosine kinase inhibitors include imatinib mesylate(Gleevec™) or tyrphostin such as Tyrphostin A23/RG-50810; AG 99;Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; TyrphostinB44; Tyrphostin B44 (+) enantiomer; Tyrphostin AG 555; AG 494;Tyrphostin AG 556, AG957 and adaphostin(4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester;NSC 680410, adaphostin); 1) compounds targeting, decreasing orinhibiting the activity of the epidermal growth factor family ofreceptor tyrosine kinases (EGFR₁ ErbB₂, ErbB3, ErbB4 as homo- orheterodimers) and their mutants, such as compounds which target,decrease or inhibit the activity of the epidermal growth factor receptorfamily are especially compounds, proteins or antibodies which inhibitmembers of the EGF receptor tyrosine kinase family, such as EGFreceptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or EGF related ligands,CP 358774, ZD 1839, ZM 105180; trastuzumab (Herceptin™), cetuximab(Erbitux™), Iressa, Tarceva, OSI-774, C1-1033, EKB-569, GW-2016, E1.1,E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3, and7H-pyrrolo-[2,3-d]pyrimidine derivatives; m) compounds targeting,decreasing or inhibiting the activity of the c-Met receptor, such ascompounds which target, decrease or inhibit the activity of c-Met,especially compounds which inhibit the kinase activity of c-Metreceptor, or antibodies that target the extracellular domain of c-Met orbind to HGF, n) compounds targeting, decreasing or inhibiting the kinaseactivity of one or more JAK family members (JAK1/JAK2/JAK3/TYK2 and/orpan-JAK), including but not limited to PRT-062070, SB-1578, baricitinib,pacritinib, momelotinib, VX-509, AZD-1480, TG-101348, tofacitinib, andruxolitinib; o) compounds targeting, decreasing or inhibiting the kinaseactivity of PI3 kinase (PI3K) including but not limited to ATU-027,SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib,pictrelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, andidelalisib; and; and q) compounds targeting, decreasing or inhibitingthe signaling effects of hedgehog protein (Hh) or smoothened receptor(SMO) pathways, including but not limited to cyclopamine, vismodegib,itraconazole, erismodegib, and IPI-926 (saridegib).

The term “PI3K inhibitor” as used herein includes, but is not limited tocompounds having inhibitory activity against one or more enzymes in thephosphatidylinositol-3-kinase family, including, but not limited toPI3Kα, PI3Kγ, PI3Kδ, P131β, PI3K-C2α, PI3K-C2β, PI3K-C2γ, Vps34, p110-α,p110-β, p110-γ, p110-δ, p85-α, p85-β, p55-γ, p150, p101, and p87.Examples of PI3K inhibitors useful in this invention include but are notlimited to ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474,buparlisib, pictrelisib, PF-4691502, BYL-719, dactolisib, XL-147,XL-765, and idelalisib.

The term “Bcl-2 inhibitor” as used herein includes, but is not limitedto compounds having inhibitory activity against B-cell lymphoma 2protein (Bcl-2), including but not limited to ABT-199, ABT-731, ABT-737,apogossypol, Ascenta's pan-Bcl-2 inhibitors, curcumin (and analogsthereof), dual Bcl-2/Bcl-xL inhibitors (InfinityPharmaceuticals/Novartis Pharmaceuticals), Genasense (G3139), HA14-1(and analogs thereof; see WO2008118802), navitoclax (and analogsthereof, see U.S. Pat. No. 7,390,799), NH-1 (Shenayng PharmaceuticalUniversity), obatoclax (and analogs thereof, see WO2004106328), S-001(Gloria Pharmaceuticals), TW series compounds (Univ. of Michigan), andvenetoclax. In some embodiments the Bcl-2 inhibitor is a small moleculetherapeutic. In some embodiments the Bcl-2 inhibitor is apeptidomimetic.

The term “BTK inhibitor” as used herein includes, but is not limited tocompounds having inhibitory activity against Bruton's Tyrosine Kinase(BTK), including, but not limited to AVL-292 and ibrutinib.

The term “SYK inhibitor” as used herein includes, but is not limited tocompounds having inhibitory activity against spleen tyrosine kinase(SYK), including but not limited to PRT-062070, R-343, R-333, Excellair,PRT-062607, and fostamatinib.

Further examples of BTK inhibitory compounds, and conditions treatableby such compounds in combination with compounds of this invention can befound in WO2008039218 and WO2011090760, the entirety of which areincorporated herein by reference.

Further examples of SYK inhibitory compounds, and conditions treatableby such compounds in combination with compounds of this invention can befound in WO2003063794, WO2005007623, and WO2006078846, the entirety ofwhich are incorporated herein by reference.

Further examples of PI3K inhibitory compounds, and conditions treatableby such compounds in combination with compounds of this invention can befound in WO2004019973, WO2004089925, WO2007016176, U.S. Pat. No.8,138,347, WO2002088112, WO2007084786, WO2007129161, WO2006122806,WO2005113554, and WO2007044729 the entirety of which are incorporatedherein by reference.

Further examples of JAK inhibitory compounds, and conditions treatableby such compounds in combination with compounds of this invention can befound in WO2009114512, WO2008109943, WO2007053452, WO2000142246, andWO2007070514, the entirety of which are incorporated herein byreference.

Further anti-angiogenic compounds include compounds having anothermechanism for their activity, e.g. unrelated to protein or lipid kinaseinhibition e.g. thalidomide (Thalomid™) and TNP-470.

Examples of proteasome inhibitors useful for use in combination withcompounds of the invention include, but are not limited to bortezomib,disulfiram, epigallocatechin-3-gallate (EGCG), salinosporamide A,carfilzomib, ONX-0912, CEP-18770, and MLN9708.

Compounds which target, decrease or inhibit the activity of a protein orlipid phosphatase are e.g. inhibitors of phosphatase 1, phosphatase 2A,or CDC25, such as okadaic acid or a derivative thereof.

Compounds which induce cell differentiation processes include, but arenot limited to, retinoic acid, α- γ- or δ-tocopherol or α- γ- orδ-tocotrienol.

The term cyclooxygenase inhibitor as used herein includes, but is notlimited to, Cox-2 inhibitors, 5-alkyl substituted2-arylaminophenylacetic acid and derivatives, such as celecoxib(Celebrex™), etoricoxib, valdecoxib or a 5-alkyl-2-arylaminophenylaceticacid, such as 5-methyl-2-(2′-chloro-6′-fluoroanilino)phenyl acetic acid,lumiracoxib.

The term “bisphosphonates” as used herein includes, but is not limitedto, etridonic, clodronic, tiludronic, pamidronic, alendronic,ibandronic, risedronic and zoledronic acid. Etridonic acid is marketedunder the trade name Didronel™. Clodronic acid is marketed under thetrade name Bonefos™. Tiludronic acid is marketed under the trade nameSkelid™. Pamidronic acid is marketed under the trade name Aredia™Alendronic acid is marketed under the trade name Fosamax™. Ibandronicacid is marketed under the trade name Bondranat™. Risedronic acid ismarketed under the trade name Actonel™. Zoledronic acid is marketedunder the trade name Zometa™. The term “mTOR inhibitors” relates tocompounds which inhibit the mammalian target of rapamycin (mTOR) andwhich possess antiproliferative activity such as sirolimus (Rapamune®),everolimus (Certican™), CCI-779 and ABT578.

The term “heparanase inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit heparin sulfate degradation. The termincludes, but is not limited to, PI-88. The term “biological responsemodifier” as used herein refers to a lymphokine or interferons.

The term “inhibitor of Ras oncogenic isoforms,” such as H-Ras, K-Ras, orN-Ras, as used herein refers to compounds which target, decrease orinhibit the oncogenic activity of Ras; for example, a “farnesyltransferase inhibitor” such as L-744832, DK8G557 or R115777(Zarnestra™). The term “telomerase inhibitor” as used herein refers tocompounds which target, decrease or inhibit the activity of telomerase.Compounds which target, decrease or inhibit the activity of telomeraseare especially compounds which inhibit the telomerase receptor, such astelomestatin.

The term “methionine aminopeptidase inhibitor” as used herein refers tocompounds which target, decrease or inhibit the activity of methionineaminopeptidase. Compounds which target, decrease or inhibit the activityof methionine aminopeptidase include, but are not limited to, bengamideor a derivative thereof.

The term “proteasome inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit the activity of the proteasome. Compoundswhich target, decrease or inhibit the activity of the proteasomeinclude, but are not limited to, Bortezomib (Velcade™) and MLN 341.

The term “matrix metalloproteinase inhibitor” or (“MMP” inhibitor) asused herein includes, but is not limited to, collagen peptidomimetic andnonpeptidomimetic inhibitors, tetracycline derivatives, e.g. hydroxamatepeptidomimetic inhibitor batimastat and its orally bioavailable analoguemarimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551)BMS-279251, BAY 12-9566, TAA211, MMI270B or AAJ996.

The term “compounds used in the treatment of hematologic malignancies”as used herein includes, but is not limited to, FMS-like tyrosine kinaseinhibitors, which are compounds targeting, decreasing or inhibiting theactivity of FMS-like tyrosine kinase receptors (Flt-3R); interferon,1-β-D-arabinofuransylcytosine (ara-c) and bisulfan; and ALK inhibitors,which are compounds which target, decrease or inhibit anaplasticlymphoma kinase.

Compounds which target, decrease or inhibit the activity of FMS-liketyrosine kinase receptors (Flt-3R) are especially compounds, proteins orantibodies which inhibit members of the Flt-3R receptor kinase family,such as PKC412, midostaurin, a staurosporine derivative, SU11248 andMLN518.

The term “HSP90 inhibitors” as used herein includes, but is not limitedto, compounds targeting, decreasing or inhibiting the intrinsic ATPaseactivity of HSP90; degrading, targeting, decreasing or inhibiting theHSP90 client proteins via the ubiquitin proteosome pathway. Compoundstargeting, decreasing or inhibiting the intrinsic ATPase activity ofHSP90 are especially compounds, proteins or antibodies which inhibit theATPase activity of HSP90, such as 17-allylamino,17-demethoxygeldanamycin(17AAG), a geldanamycin derivative; other geldanamycin relatedcompounds; radicicol and HDAC inhibitors.

The term “antiproliferative antibodies” as used herein includes, but isnot limited to, trastuzumab (Herceptin™), Trastuzumab-DM1, erbitux,bevacizumab (Avastin™), rituximab (Rituxan®), PRO64553 (anti-CD40) and2C4 Antibody. By antibodies is meant intact monoclonal antibodies,polyclonal antibodies, multispecific antibodies formed from at least 2intact antibodies, and antibodies fragments so long as they exhibit thedesired biological activity.

For the treatment of acute myeloid leukemia (AML), compounds of thecurrent invention can be used in combination with standard leukemiatherapies, especially in combination with therapies used for thetreatment of AML. In particular, compounds of the current invention canbe administered in combination with, for example, farnesyl transferaseinhibitors and/or other drugs useful for the treatment of AML, such asDaunorubicin, Adriamycin, Ara-C, VP-16, Teniposide, Mitoxantrone,Idarubicin, Carboplatinum and PKC412.

Other anti-leukemic compounds include, for example, Ara-C, a pyrimidineanalog, which is the 2′-alpha-hydroxy ribose (arabinoside) derivative ofdeoxycytidine. Also included is the purine analog of hypoxanthine,6-mercaptopurine (6-MP) and fludarabine phosphate. Compounds whichtarget, decrease or inhibit activity of histone deacetylase (HDAC)inhibitors such as sodium butyrate and suberoylanilide hydroxamic acid(SAHA) inhibit the activity of the enzymes known as histonedeacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228(formerly FR901228), Trichostatin A and compounds disclosed in U.S. Pat.No. 6,552,065 including, but not limited to,N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide,or a pharmaceutically acceptable salt thereof andN-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide,or a pharmaceutically acceptable salt thereof, especially the lactatesalt. Somatostatin receptor antagonists as used herein refer tocompounds which target, treat or inhibit the somatostatin receptor suchas octreotide, and SOM230. Tumor cell damaging approaches refer toapproaches such as ionizing radiation. The term “ionizing radiation”referred to above and hereinafter means ionizing radiation that occursas either electromagnetic rays (such as X-rays and gamma rays) orparticles (such as alpha and beta particles). Ionizing radiation isprovided in, but not limited to, radiation therapy and is known in theart. See Hellman, Principles of Radiation Therapy, Cancer, in Principlesand Practice of Oncology, Devita et al., Eds., 4^(th) Edition, Vol. 1,pp. 248-275 (1993).

Also included are EDG binders and ribonucleotide reductase inhibitors.The term “EDG binders” as used herein refers to a class ofimmunosuppressants that modulates lymphocyte recirculation, such asFTY720. The term “ribonucleotide reductase inhibitors” refers topyrimidine or purine nucleoside analogs including, but not limited to,fludarabine and/or cytosine arabinoside (ara-C), 6-thioguanine,5-fluorouracil, cladribine, 6-mercaptopurine (especially in combinationwith ara-C against ALL) and/or pentostatin. Ribonucleotide reductaseinhibitors are especially hydroxyurea or2-hydroxy-1H-isoindole-1,3-dione derivatives.

Also included are in particular those compounds, proteins or monoclonalantibodies of VEGF such as1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceuticallyacceptable salt thereof,1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate;Angiostatin™; Endostatin™; anthranilic acid amides; ZD4190; Zd₆474;SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGFreceptor antibodies, such as rhuMAb and RHUFab, VEGF aptamer such asMacugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibody,Angiozyme (RPI 4610) and Bevacizumab (Avastin™)

Photodynamic therapy as used herein refers to therapy which uses certainchemicals known as photosensitizing compounds to treat or preventcancers. Examples of photodynamic therapy include treatment withcompounds, such as Visudyne™ and porfimer sodium.

Angiostatic steroids as used herein refers to compounds which block orinhibit angiogenesis, such as, e.g., anecortave, triamcinolone,hydrocortisone, 11-α-epihydrocotisol, cortexolone,17α-hydroxyprogesterone, corticosterone, desoxycorticosterone,testosterone, estrone and dexamethasone.

Implants containing corticosteroids refers to compounds, such asfluocinolone and dexamethasone.

Other chemotherapeutic compounds include, but are not limited to, plantalkaloids, hormonal compounds and antagonists; biological responsemodifiers, preferably lymphokines or interferons; antisenseoligonucleotides or oligonucleotide derivatives; shRNA or siRNA; ormiscellaneous compounds or compounds with other or unknown mechanism ofaction.

The structure of the active compounds identified by code numbers,generic or trade names may be taken from the actual edition of thestandard compendium “The Merck Index” or from databases, e.g. PatentsInternational (e.g. IMS World Publications).

A compound of the current invention may also be used in combination withknown therapeutic processes, for example, the administration of hormonesor radiation. In certain embodiments, a provided compound is used as aradiosensitizer, especially for the treatment of tumors which exhibitpoor sensitivity to radiotherapy.

A compound of the current invention can be administered alone or incombination with one or more other therapeutic compounds, possiblecombination therapy taking the form of fixed combinations or theadministration of a compound of the invention and one or more othertherapeutic compounds being staggered or given independently of oneanother, or the combined administration of fixed combinations and one ormore other therapeutic compounds. A compound of the current inventioncan besides or in addition be administered especially for tumor therapyin combination with chemotherapy, radiotherapy, immunotherapy,phototherapy, surgical intervention, or a combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies, as described above. Otherpossible treatments are therapy to maintain the patient's status aftertumor regression, or even chemopreventive therapy, for example inpatients at risk.

Those additional agents may be administered separately from a disclosedX4P-001 composition, as part of a multiple dosage regimen.Alternatively, those agents may be part of a single dosage form, mixedtogether with a disclosed X4P-001 composition in a single composition.If administered as part of a multiple dosage regime, the two activeagents may be submitted simultaneously, sequentially or within a periodof time from one another normally within five hours from one another.

As used herein, the term “combination,” “combined,” and related termsrefers to the simultaneous or sequential administration of therapeuticagents in accordance with this invention. For example, a compound of thepresent invention may be administered with another therapeutic agentsimultaneously or sequentially in separate unit dosage forms or togetherin a single unit dosage form. Accordingly, the present inventionprovides a single unit dosage form comprising a compound of the currentinvention, an additional therapeutic agent, and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle.

The amount of both a disclosed X4P-001 composition and additionaltherapeutic agent (in those compositions which comprise an additionaltherapeutic agent as described above) that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration.Preferably, compositions of this invention should be formulated so thata dosage of between 0.01-100 mg/kg body weight/day of an inventivecompound can be administered.

In those compositions which comprise an additional therapeutic agent,that additional therapeutic agent and the compound of this invention mayact synergistically. Therefore, the amount of additional therapeuticagent in such compositions will be less than that required in amonotherapy utilizing only that therapeutic agent. In such compositionsa dosage of between 0.01-1,000 μg/kg body weight/day of the additionaltherapeutic agent can be administered.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed X4P-001 compositions will range from about50% to 100% of the amount normally present in a composition comprisingthat agent as the only therapeutically active agent.

The compounds of this invention, or pharmaceutical compositions thereof,may also be incorporated into compositions for coating an implantablemedical device, such as prostheses, artificial valves, vascular grafts,stents and catheters. Vascular stents, for example, have been used toovercome restenosis (re-narrowing of the vessel wall after injury).However, patients using stents or other implantable devices risk clotformation or platelet activation. These unwanted effects may beprevented or mitigated by pre-coating the device with a pharmaceuticallyacceptable composition comprising a kinase inhibitor. Implantabledevices coated with a compound of this invention are another embodimentof the present invention.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Exemplification

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein.

Methods of preparation applicable to certain compounds of the inventionare disclosed in Crawford et al. (2008) Org. Process Res. Dev.12:823-830; U.S. Pat. No. 7,354,934, WO 00/56729, U.S. Ser. No.60/232,891, and U.S. Ser. No. 60/234,510, as well as An, H.; Wang, T.;Mohan, V.; Griffey, R. H.; Cook, P. D. Tetrahedron 1998, 54, 3999-4012;the entire contents of each of which is hereby incorporated byreference. One of ordinary skill in the art is capable of varying suchdisclosed methods, using no more than routine experimentation, toprovide alternate means of preparation, testing, and analysis ofcompounds of the invention.

Example 1: Methyl Imine Impurity

By high performance liquid chromatography-mass spectrometry (HPLC-MS)analysis, a peak at relative retention time (RRT) 1.13 showed a [M+1]+of 362 m/z (12 mass units higher than X4P-001). The peak disappearsafter a few hours if X4P-001 is solubilized in acidic aqueous media. Themolecular weight of the impurity along with its chemical behaviorsuggest a methyl imine structure on the primary amine. The imineimpurity, I-1, and likely route of formation are shown in Scheme 1,below.

Example 2: N-Formyl Impurity

By HPLC-MS analysis a RRT 1.28 peak showed an [M+1]+ of 378 m/z (28 massunits higher than X4P-001), which suggested an N-formyl derivative ofX4P-001. This has been confirmed by an independent synthesis. Directreaction of X4P-001 with ethyl formate generated the N-formyl compoundI-2, which is identical to the batch impurity based on MS and HPLC data.Formation of the formate is shown in Scheme 2, below.

Example 3: Aldehyde Impurity

A RRT 1.14 peak was characterized as I-6. Without wishing to be bound byany particular theory, it is believed that I-6 results from oxidation ofthe amine on the alkyl chain of X4P-001, as shown in Scheme 3, below:

Example 4: Acetamide Impurity

By HPLC-MS analysis, a RRT 1.39 peak showed an [M+1]+ of 392 m/z (42mass units higher than X4P-001), which was characterized as the X4P-001acetamide. It is formed at elevated temperatures by reaction of X4P-001with the isopropyl acetate crystallization solvent as shown in Scheme 4.An independent synthesis of the acetamide impurity confirmed thestructure. Reaction of X4P-001 with acetic anhydride gave the acetamideI-5, which is identical to the batch impurity, based on the MS and HPLCdata. Isopropyl acetate is used for certain methods of manufacture ofX4P-001 free base that use the p-hydroxy benzoic acid salt of X4P-001.

Example 5: Benzimidazole Impurity

By LC-MS analysis, a RRT 1.67 peak showed an [M+1]+ of 481 m/z (131 massunits higher than X4P-001), which was characterized as theM+benzimidazole compound I-3. It is formed as an impurity during thet-butoxycarbonyl (Boc group) protection of 2-chloromethylbenzimidazoleas shown in Scheme 5, below. During the N-alkylation reaction (step 2 ofthe X4P-001 synthesis), the impurity undergoes alkylation and as aconsequence is present throughout the remainder of the manufacturingprocess.

An independent synthesis, involving reaction of a secondary amine 2918with boc-2-chloromethylbenzimidazole 2890 (I-9), followed bydeprotection afforded I-3, which was identical to the observed impuritybased on MS and HPLC.

Example 6: Aminal Impurity

An aminal impurity arises in the production of the p-hydroxybenzoic acidsalt of X4P-001 by the reaction of the imine impurity withp-hydroxybenzoic acid. This impurity is relevant to the manufacture ofX4P-001 in methods that use ap-hydroxybenzoic acid salt of X4P-001 orintermediates thereof.

The structure of the aminal impurity I-4 and its origin are shown belowin Scheme 6.

Example 7: Basis for Setting the Acceptance Criteria for OrganicImpurities

The impurity results for relevant batches of X4P-001 and its p-hydroxybenzoic acid (PHB) salt are shown in Table 2, below. The X4P-001 batchesused in initial toxicology studies were primarily used in setting theimpurity specifications. Batches of X4P-001 p-hydroxy benzoic acid saltwere used in longer term toxicology studies and have also been presentedin Table 2. Total impurities in these six batches ranged from <0.05% to1.4% with individual unspecified impurities >0.05% present in only asingle batch at the 0.1% level. Taking into account the limited numberof batches produced and the impurity profiles of the X4P-001 batchesused for toxicology and clinical studies, the drug substance releasespecification for total impurities has been set at <3.0% w/w with nosingle unspecified impurity greater than 0.2% w/w (0.5% for stability)(Table 2). Specified individual impurities and p-hydroxy benzoic acidcontent are all <0.5% w/w except for the imine impurity which has beenset at 1.1% based on the level of this impurity administered intoxicology studies. A separate batch of product (not shown in Table 2),namely X4P-001 batch 3-1 (Table 4), provided the drug substance in highpurity with total impurities being 1.20% w/w and no specified impuritybeing greater than 0.07% w/w except for the imine impurity, which was0.62% w/w. The assay result of the clinical X4P-001 drug substance batchwas 99.3% w/w and the chiral purity was >99% ee.

TABLE 2 Batch Number 2-1 2-2 2-3 2-4 2-5 2-6 2-7 Drug Free Free FreeFree Free PHB PHB Substance Base Base Base Base Base salt salt TypeIndividual Results of Testing Impurities by HPLC (% w/w) on anhydrousbasis Imine (I-1) 1.1 0.5 0.1 0.49 0.65 0.02 0.02 Formyl (I-2) 0.2 0.10.1 0.13 0.13 ND ND Acetamide 0.1 0.1 0.1 0.10 0.06 NA NA (I-5) Aminal(I-4) NA NA NA NA NA 0.01 ND Benzimida- 0.01^(a) NR 0.2 <LOQ 0.11 0.010.01 zole (I-3) Single NR NR 0.1 ND NR ND ND Unspecified PHB NA NA NA NANA 28.1 28.4 Content Total 1.4 0.7 0.5 0.72 1.01 ≤0.05 ≤0.05 Impurities(% w/w)^(b) (R)- >99 >99 >99 >99 >99 >99 >99 enantiomer % ee by HPLCAbbreviations used: ^(a)Value below LOQ (limit of quantitation)^(b)Specification only relevant for free base clinical drug substancemanufactured using X4P-001 p-hydroxybenzoic acid salt as the startingmaterial NA = Not Applicable ND = None Detected NLT = Not Less Than NMT= Not More Than NR = None Reported

Typically, the wt % of each impurity is determined by HPLC, and ismeasured either initially or after storage, and optionally on anon-going basis during the shelf life of the X4P-001 composition. In someembodiments, the level of an impurity is measured after storage of thecomposition under stressed conditions, which are conditions of elevatedtemperature, humidity, or both, used to approximate the effect oflong-term storage under ambient conditions. Accordingly, in someembodiments, the present invention provides an X4P-001 compositioncomprising no more than 1.1% imine (I-1); no more than 0.3% formyl(I-2); no more than 0.4% benzamidazole (I-3); no more than 0.5% aminal(I-4); no more than 0.5% acetamide (I-5); no more than 0.4% aldehyde(I-6); no more than 0.3% de-BOC NT-316 (I-7); and no more than 0.2% of asingle unspecified impurity. Additionally, when free base clinical drugsubstance is manufactured via X4P-001 p-hydroxybenzoic acid salt, nomore than 1.0% PHB is present in the composition. In some embodiments,no more than 3.0% of total impurities are present. In some embodiments,the R-enantiomeric enantiomeric excess (% ee) is no less than 97.0%.

Example 8: Identification of Impurities Resulting after Storage

Upon analysis of stability trial samples of X4P-001 (PTL/ST/0511) byMET/CR/1448 stored at 25° C./60% Relative Humidity (25/60) for threemonths showed the formation of two unknown impurities.

-   -   Unknown 1—RRT 1.14    -   Unknown 2—RRT 1.24

The impurity at RRT 1.14 was also the principal degradation productunder elevated temperature and humidity (80° C./80% RH) conditions inPTL/DA/0175.

SUMMARY

Two impurities formed during a stability study on X4P-001 at t=3 monthsat 25° C./60% RH and in a forced degradation study under elevatedtemperature/humidity were identified by LC-MS.

Experimental

Instrument Parameters

Work was performed on a GMP Waters Alliance HPLC system connected to aZQ 2000 single quad MS. Data were collected into Empower 2 software.

The assay and impurities method for X4P-001 (MET/CR/1448) is not ideallysuited for mass spectrometry because it uses TFA as a modifier in theeluent which can signifcantly suppress ionisation. Initially this wassubstituted for 0.15% v/v formic acid. The flow rate was also changed toallow connection directly into the inlet of the MS (maximum flow 0.3mL/min) and the gradient adjusted accordingly.

HPLC Conditions 1

-   -   Column: Zorbax Bonus-RP, 150×4.6 mm, 3.5 μm    -   Inj. volume: 100 μL    -   Detection: UV @ 220 nm (190-400 nm)        -   MS ES+100-700 Da, ES-100-700 Da    -   Mobile phase A: 0.15% formic acid in Water    -   Mobile phase B: 0.15% formic acid in MeCN

Time (mins) % A % B 0.0 95 5 6 95 5 51 5 95 60 5 95 60.1 95 5 75 95 5

-   -   Flow rate: 0.3 mL/min    -   Column temperature: Ambient    -   Run time: 75 minutes

MS Tune Parameters 1 ES+ ES− Ion source tab Capillary Voltage (kV) 3.5 4Cone Voltage (V) 25 25 Extractor (V) 3 0 RF lens 0.4 3 Source Temp ° C.150 150 Desolvation Temp ° C. 150 150 Desolvation gas flow (L/hr) 300300 Cone gas flow (L/hr) 50 50 Analyzer tab LM Res 15 15 HM Res 15 15Ion Energy 1.8 2 Multiplier 521 521

X4P-001 was not retained under these conditions because it required theion-pairing of TFA. Chromatography and mass spectra obtained arepresented in FIG. 5.

Tentative masses were obtained for two impurities. All mass spectrapresented have been background corrected (region taken immediatelybefore and after the peak of interest.

-   -   Unknown 1 MET/CR/1448 RRT 1.14 [M+H]=m/z 349.2    -   Unknown 2 MET/CR/1448 RRT 1.24 [M+H]=m/z 392.3

The inventors hypothesized that there was a possibility that theimpurities of interest were co-eluting with other impurities. Sampleswere therefore run using the chromatographic conditions in MET/CR/1448.Concentration and injection volumes were increased to counteractsuppression of ionization caused by the TFA.

HPLC Conditions 2

-   -   Column: Zorbax SB-C8, 150 mm×4.6 mm, 3.5 μm    -   Guard column: Zorbax SB-C8, 12.5 mm×4.6 mm, 5 μm    -   Inj. volume: Various    -   Detection: UV @ 270 nm    -   Mobile phase: Mobile phase A: 0.2% TFA in water        -   Mobile phase B: 0.1% TFA in acetonitrile

Gradient elution Time Flow rate (mins) % A % B (mL/min) 0 92 8 0.8 5 9010 0.8 15 89 11 0.8 25 80 20 0.8 28 80 20 0.8 37 55 45 0.8 44 20 80 0.847 20 80 0.8 48 92 8 1.2 53 92 8 1.2 54 92 8 0.8 55 92 8 0.8

Initial flow rate: 0.8 mL/min (refer to gradient timetable) split 4:1into the MS inlet.

-   -   Column temperature: 25° C.    -   Run time: 55 minutes

MS parameters as per Tune Parameters 1 above.

Various samples of X4P-001 in which the impurities of interest had beendetected were prepared in methanol at either 1 or 10 mg/mL:

-   -   PTL/ST/0511, batch 3-1, 25/60 t=3 months    -   PTL/DA/0175 degradation sample 80° C./80% relative humidity        (80/80) t=1 and 7 days.

Data obtained are presented in FIGS. 6-9.

Mass spectral data obtained from HPLC conditions 2 were confirmed:

-   -   Unknown 1 MET/CR/1448 RRT 1.14 [M+H]=m/z 349.2    -   Unknown 2 MET/CR/1448 RRT 1.24 [M+H]=m/z 392.3

Finally, the chromtographic conditions were modified to allow the flowto enter the MS without being split. Results from HPLC Conditions 3 areshown in FIG. 10.

HPLC Conditions 3

-   -   Column: Zorbax SB-C8, 150 mm×4.6 mm, 3.5 μm    -   Guard column: Zorbax SB-C8, 12.5 mm×4.6 mm, 5 μm    -   Inj. volume: Various    -   Detection: UV @ 270 nm    -   Mobile phase: Mobile phase A: 0.2% TFA in water        -   Mobile phase B: 0.1% TFA in acetonitrile

Gradient elution Time (mins) % A % B 0 92 8 13.3 90 10 39.9 89 11 66.5080 20 74.48 80 20 98.42 55 45 117 20 80 125 20 80 125.10 92 8 140 92 8

-   -   Flow rate: 0.3 mL/min directly into the MS inlet    -   Column temperature: 25° C.    -   Run time: 140 minutes    -   MS parameters as per Tune Parameters 1 above.

Discussion of Results

All of the spectra obtained confirmed the initial results even thoughpeak responses and resolution from X4P-001 varied.

-   -   Unknown 1 MET/CR/1448 RRT 1.14 [M+H]=m/z 349.2    -   Unknown 2 MET/CR/1448 RRT 1.24 [M+H]=m/z 392.3

Unknown 1 RRT 1.14 (Aldehyde)

The molecular weight of 348 indicates the loss of a nitrogen (nitrogenrule). This is consistent with the oxidation of the amine on the alkylchain yielding the aldehyde. The postulated structure is shown below.

-   -   Empirical formula=C21H24N4O    -   Monoisotopic mass=348.195

Unknown 2 RRT 1.24 (Acetamide)

The molecular weight of 391 is consistent with I-5, the acetamideimpurity (formed with the reaction of X4P-001 with residual isopropylacetate). The structure of I-5 is shown below.

Conclusions

Both unknown impurities were identified from the mass specta dataobtained. The impurity at RRT 1.14 is an aldehyde oxidation product andthe impurity at RRT 1.24 is the acetamide impurity I-5.

Example 9: Improvements to Manufacturing Process for X4P-001 to Reduceand Control Impurity Levels Introduction

The final step in the previous preparation of GMP drug substance insupport of clinical studies (process version 2; shown in FIG. 2) startedwith compounds NT-316 and AMD-2890 (I-9). After studying the structureand origin of the most significant impurities which were formed in thispreparation, a new process (process version 3) has been developed whichoffers improved control over the impurity profile as well as thecrystallization of the API. Moreover, this effort has significantlyenhanced the reproducibility and robustness of the final step in theprocess.

Description of Synthetic Route and Key Intermediates

API X4P-001 is assembled in 2 steps in a convergent manner starting fromtwo key fragments: NT-316 and AMD-2890. The penultimate intermediateAMD-11070 is not isolated but immediately converted to the API, which iscrystallized after a work-up procedure.

Scheme 7 and Scheme 8 show the synthesis for the API molecule X4P-001.

Key fragment AMD-2890 is synthesized in one step from RM-396 (I-8). Eventhough RM-396 is commercially available, its purity is highly variabledepending on its source. The purity of commercially sourced material canappear to be high (>97 area %) according to HPLC analysis, however thetrue wt % purity has been found to be as low as 90%. It is likely thatRM-396 undergoes self-alkylation, resulting in the formation of avariable mixture of oligomeric impurities. Only after conversion ofRM-396 into AMD-2890 is a stable compound with a well-defined impurityprofile obtained.

The other key fragment is NT-316, a stable, crystalline andwell-characterized compound that incorporates a significant part of theAPI structure. NT-316 is obtained by a convergent synthesis via reactionof NT-319 with the chiral amine salt NT-272. NT-272 is a stablecrystalline compound, which can be procured via custom synthesis.

NT-319 is a highly unstable intermediate which cannot be isolatedwithout degradation. It is prepared in 3 steps (each without isolationof intermediates) from commercial ABA. Control of the purity of ABA isdifficult because it is not only an oil, but it also lacks a UVchromophore, severely limiting the methods for its precise analyticalcharacterization.

On the basis of the above, it is the impurity profile of these twofragments NT-316 and AMD-2890 (I-9), in combination with the exactconditions of the two final chemical steps (which are to be conductedunder GMP control), that determine the impurity profile and quality ofthe API.

In summary, the chemical and enantiomeric purity of NT-316 and thechemical purity of AMD-2890 can be directly correlated with the chemicaland enantiomeric purity of the API (X4P-001).

Description of Key Process Changes

FIG. 3 shows how the new process (Process 3) differs from the previousversion (Process 2). In process version 2, it proved necessary toisolate the API first as its 4-hydroxybenzoate salt. This salt was thenconverted to the corresponding free base in a separate step in order toobtain the API.

The chemical steps in process version 3 are unchanged. Compounds NT-316and AMD-2890 (I-9) are reacted with each other and the resulting product(Tri-Boc), which is not isolated but rather deprotected immediately toafford the crude API. However, the method utilized for isolating the APIhas changed considerably in process version 3.

Among the most significant impurities that are produced in processversion 2 are imine A (RRT 1.08) and N-formyl B (RRT 1.28), as shown inScheme 9. It was shown that these impurities originated from the use ofdichloromethane as a solvent during the extractions of the crude API.Furthermore, formation of acetamide impurity C (RRT 1.37) could becorrelated with the use of isopropyl acetate as the solvent for thefinal crystallization of the API free base.

Thus, dichloromethane and isopropyl acetate, which were used in thework-up and isolation of X4P-001 in version 2, were replaced with1-butanol and a toluene/methanol mixture, respectively, for version 3.We have found that these solvents do not react with the API, andaccordingly we believe that this change has caused the significantreduction of impurities A (imine), B (N-formyl) and C (acetamide) thatwe have ob served.

In addition, it was found that problems with formation of gums andoiling of the API during the final product crystallization in processversion 2 were due to the quality of the AMD-2890 starting material.This material is obtained from the commercial2-chloromethylbenzimidazole (RM-396), which can have a low wt % purityeven when the area % purity appears to be good (>98%). The developmentof an improved isolation procedure for AMD-2890 is an integral part ofprocess version 3 and this has consistently resulted in the productionof high quality AMD-2890, which possesses not only excellent % puritybut also assay % w/w purity.

Further optimization of the aeration and carbon treatments during theisolation of the API has resulted in better control over the color ofthe isolated API and these operations are also included in processversion 3.

Finally, the crystallization of the free base API from toluene wasnarrowly defined. The meta-stable zone and the optimum seeding pointwere determined. We also developed an optimum cooling rate afterformation of the seed bed, as well as an appropriate washing and dryingprotocol.

FIG. 4 provides a detailed comparison between Process version 2 and 3for the consecutive downstream operations in the work-up and isolationof the API.

The robustness and reproducibility of the process version 3 wasdemonstrated through the performance of three identical lab experimentson 100 g scale. As the data in Table 3 and Table 4 show, this processdemonstration was successful. We subsequently scaled up the process to a10 kg scale. A total of 9.75 kg of GMP X4P-001 was prepared in a singlebatch with nearly identical outcomes. The details of these experimentsare discussed below.

Description of Process Version 3 and Improvements of Process 1 andProcess 2

The following section provides a brief description of the variousoperations in the synthesis and isolation of the API in the final stepof process version 3.

Synthesis of NT-316

The early process steps for the preparation of the key starting materialNT-316 from the custom synthesized chiral amine NT-272 and ABA areunchanged from earlier campaigns.

Synthesis of AMD-2890

As noted above, the synthesis AMD-2890 (I-9) has been improved, inparticular its isolation and crystallization, to accommodate for thevariable purity and color of commercial 2-chloromethylbenzimidazole(RM-396). This material is typically dark brown to black. A charcoaltreatment was therefore introduced to allow better control of the colorof AMD-2890.

Thus, 2-chloromethylbenzimidazole (RM-396) is reacted with 1.3 equiv. ofdi-tert-butyl dicarbonate in DMF (N,N-dimethylformamide) 8.6 volumes(vol) at 40° C. in the presence of 0.1 equivalent (equiv.) of DIPEA(diisopropylethylamine). After completion of the reaction decolorizingcharcoal is added. After aging for 1.5 h at 40° C. the mixture isfiltered, and the solids washed with 1 vol of DMF. Water (3.5 vol) isadded slowly to the filtrates, resulting in a slightly turbid mixturewhich is seeded with 1% of AMD-2890. Upon aging, a slurry is producedwhich is allowed to cool slowly to 20° C. After slow addition of morewater (1 vol) and further cooling to 0° C. the slurry is filtered. Thesolids are first washed with a 2:1 mixture of DMF and water, then withwater (2×3 vol), all at 0° C. The filter cake is dried under a stream ofnitrogen providing the light-yellow AMD-2890. This compound istypically >99 area % (by HPLC) and >99 wt % (by NMR) pure.

Synthesis and Isolation of X4P-001

As before, equimolar quantities of NT-316 and AMD-2890 are reacted inthe presence of diisopropylethylamine (DIPEA) and tetrabutylammoniumiodide (TBAI) in acetonitrile at 60-65° C. Once complete, the reactionis cooled to ambient temperature and quenched with 0.3 vol of ammoniabefore 1 vol of water is added. The resulting mixture is then added tomixture of 2 vol of concentrated hydrochloric acid and 3 vol of water.Aging for several hours at 35-40° C. in this acidic solution effects thedeprotection of the protected intermediate to the API. The acetonitrileis subsequently removed via a vacuum distillation.

At this point, 2 vol of 1-butanol are added and the pH is adjusted to 12with 20% NaOH solution. The resulting biphasic mixture is sparged with amixture of 10% of oxygen in nitrogen for 2 h at 20° C. The pH issubsequently adjusted to 3.0-3.5 with a solution of 18% HCl in water.The resulting two phases are separated, and the aqueous layer is washedwith 1-butanol (3×3 vol). The organic layers are combined and extractedwith 3 vol of water. All aqueous phases are then combined and 0.4% (wt)of carbon is added. After I-2 h the mixture is filtered, and the solidswashed with 3 vol of water.

Toluene (7 vol) and methanol (1 vol) are added to the combined filtratesand the temperature is increased to 45-55° C. before the pH is adjustedto 9.5-10.0 with 20% sodium hydroxide solution. The phases areseparated, and the aqueous layers is extracted twice more with 3 vol oftoluene.

All toluene layers are combined and partially concentrated via vacuumdistillation at 45-50° C. After several additions of fresh toluene andcontinued vacuum distillation at 45-50° C. to remove other volatilesolvents a solution of the API in approximately 3 vol of toluene isobtained. This solution is heated to 60° C. and cleared via linefiltration.

Upon cooling to 50° C. this solution is carefully seeded with up to 0.5wt % of X4P-001. After a seedbed develops a steady crystallization ofthe API is allowed to occur over 2-3 h before the slurry is allowed tocool slowly to 0° C. The resulting slurry is gradually warmed back to30-35° C. and then allowed to cool back to 0° C. to promote crystalgrowth. At the end the slurry is filtered, and the final solids arewashed with toluene and dried in a filter dryer via heating under vacuumat 60° C. for 16 h, with occasional careful agitation.

This procedure has been demonstrated on 10 kg scale which providedX4P-001 with 99.0-99.5 area % purity (>99.9% enantiopurity) and 1337 ppmof residual toluene.

Comparison of Process 2 and Process 3

Table 3 provides a comparison of key process parameters and outcomes forbatches produced most recently with Process 2 and 3. As mentioned above,process version 3 was first demonstrated on lab scale and subsequentlyscaled up in the plant for production of X4P-001. Table 4 provides acomparison of the impurity profiles of these batches.

Most importantly, Table 4 shows that a switch from process version 2 toprocess version 3 has consistently resulted in an overall 0.5-1.1 area %reduction in total impurities as well as a significant reduction intotal residual solvents in the API with no adverse impacts on othercritical process outcomes.

TABLE 3 Comparison of Key Process Parameters and Outcomes of BatchesProduced with Process Versions 2 and 3. Batch number 3-1 3-2 3-3 3-4 3-53-6 Process version 2 2 3 3 3 3 Appearance Pale yellow Pale yellow Paleyellow Pale yellow Pale yellow Pale yellow solid solid solid solid solidsolid Chiral purity >99% >99% >99.9% >99.9% >99.9% >99.9% Heavy metals<20 ppm <20 ppm <5 ppm <5 ppm <5 ppm Not performed Residual solventsiPAc 4148 ppm iPAc 7440 ppm iPAc ND iPAc ND iPAc ND iPAc ND (by GC) DCM8 ppm DCM ND DCM ND DCM ND DCM ND DCM ND IPA 6 ppm IPA 52 ppm IPA 65 ppmIPA ND IPA 109 ppm IPA ND Tol ND Tol ND Tol 2020 ppm Tol 3022 ppm Tol2020 ppm Tol 1337 ppm Moisture (by KF) <0.1 wt % 0.2 wt % 0.2 wt % 0.2wt % 0.3 wt % 0.2 wt % PSD (by laser D(50) 140 μm D(50) 118 μm D(50) 91μm D(50) 169 μm D(50) 122 μm D(50) 48 μm diffraction) ND = Not detected;iPAc = isopropyl acetate; DCM = dichloromethane; IPA = isopropylalcohol; Tol = toluene.

TABLE 4 Comparison of HPLC Impurity Profile of Batches Produced withProcess Version 2 and 3. Batch number 3-1 3-2 3-3 3-4 3-5 3-6 Processversion 2 2 3 3 3 3 Assay (wt %, oab) 99.3 97.9 99.9 96.7 97.8 98.4Purity (%) 98.8 98.3 99.46 99.40 99.28 99.51 Identified impurities (%)RRT 0.38 (de-Boc NT-316) (I-7) ND ND 0.11 0.09 0.13 0.05 RRT 1.08(imine) (I-1) 0.62 0.95 0.10 0.13 0.04 0.09 RRT 1.21 (aldehyde) (I-6) NT*  NT* 0.09 0.14 0.20 0.14 RRT 1.28 (formyl) (I-2) 0.03 ND 0.03 0.030.03 0.05 RRT 1.37 (acetamide) (I-5) 0.07 0.42 0.02 0.04 0.06 ND RRT1.93 (benzimidazole) (I-3) 0.01 ND 0.06 0.05 0.04 0.04 Total impurities(%) 1.20 1.67 0.54 0.60 0.72 0.49 ND = Not detected. NT = Not tested.*For these two batches, it is unknown whether any aldehyde impurity waspresent in these batches.

Improvements in Residual Solvent Levels in Process 3

During early clinical development of X4P-001 (formerly AMD110170), thedrug substance was directly isolated as the freebase using ethyl acetateas a crystallization solvent (Process 1). However, difficulties wereencountered in removing the ethyl acetate from the final API, withdevelopment lots requiring laborious efforts, including grinding andexposure to hot nitrogen, in order to reduce the levels below theInternational Conference on Harmonisation (ICH) limit of NMT 5000 ppm.During development of AMD-110170, early efforts also employed isopropylacetate in several batches of API as the isolation solvent in Process 1.

Later in development, the parahydroxybenzoate (PHB) salt of AMD-11070was isolated as an intermediate, followed by conversion to the freebase(Process 2). We have used this process to manufacture drug substance forrecent clinical trials. In Process 2, the freebase was isolatedfollowing salt release using isopropyl acetate instead of ethyl acetate.This API displayed similarly high levels of residual solvents as seen inAPI made via Process 1. Additionally, we have found that isopropylacetate has been implicated in the generation of the acetamide impurityduring production. Therefore, a change in the final API isolationsolvent was warranted.

Our recent process development has afforded a direct free base isolationprocedure (PHB salt is no longer an intermediate in the process) whichnow employs toluene instead of isopropyl acetate as the finalcrystallization solvent (Process 3). We found that toluene does notreact with X4P-001, and therefore provides a more appropriate solventfor isolation. As noted above, eliminating use of isopropyl acetate alsoleads to a reduction in impurity levels. A summary of residual solventlevels in batches of X4P-001, and their residual crystallization solventlevels, is presented in Table 5.

TABLE 5 Residual Isolation Solvent Levels for X4P-001 During DevelopmentMeasured Levels Isolation Solvent (ppm) Process #1 Ethyl or Isopropylacetate 7219 Ethyl or Isopropyl acetate 9108 Ethyl or Isopropyl acetate1971 Ethyl or Isopropyl acetate 6820 Ethyl or Isopropyl acetate 9298Process #2 Isopropyl acetate 2500 Isopropyl acetate**  4148** Isopropylacetate 7440 Isopropyl acetate & Toluene* ND (iPAc)* 2280 (Toluene)*Process #3 Toluene 4487 Toluene 2020 Toluene 3022 Toluene 2020 Toluene1337 *API batch was reworked from a batch originally crystallized usingisopropyl acetate, using toluene at the final crystallization stage,therefore this lot was assayed for both isopropyl acetate (iPAc) andtoluene. **Batch 3-1.

Historically, batches of X4P-001 have shown average residual solventlevels of the primary crystallization solvent in the range of 1971 ppmto 9298 ppm. We have employed the PDE approach to set a specificationfor residual toluene in X4P-001 freebase.

Assuming a dose of 600 mg X4P-001 per day (a 50% safety margin over anexemplary clinical dose of 400 mg per day), the calculated levels oftoluene in X4P-001 API would be not more than 4500 ppm. Therefore, incertain embodiments of the present invention, the specification forresidual toluene in X4P-001 is not more than 4500 ppm. Accordingly, insome embodiments, the present invention provides an X4P-001 compositioncomprising no more than 4500 ppm of toluene or 1350 ppm of toluene.

Conclusions

Taken together, the above improvements in Process version 3 haveresulted in a more robust and reproducible final process step whencompared with version 2. Most importantly, the purity of the isolatedAPI X4P-001 has significantly improved when compared with the previousprocess version. Importantly, we have discovered that the choice ofsolvents in the work-up and isolation of X4P-001 is key to the improvedimpurity profile described above. Specifically, we have replaceddichloromethane and isopropyl acetate, which were used in the work-upand isolation of X4P-001 in Process version 2, with 1-butanol and atoluene/methanol mixture, respectively, in Process version 3. We havefound that, in batches produced using Process version 2, certain of theimpurities are present in increased amounts in the final product due tothe API, X4P-001, reacting with dichloromethane and isopropyl acetate.Another key discovery was that the formation of gums and oiling of theAPI during the final product crystallization in process version 2 weredue to the variable (and often poor) quality of the AMD-2890 startingmaterial. As described above, we have introduced an improved isolationprocedure for AMD-2890 that consistently produces high quality AMD-2890and avoids the formation of gums and oiling of the API.

Example 10: Mutagenicity Evaluation

We have performed a Mutagenic Risk Assessment on the synthetic processto generate X4P-001, all process intermediates, potential and actualimpurities. The assessment included review of all raw materials,including key starting materials, as well as the potential and actualprocess impurities and degradation products. All assessments werecarried out in accordance with ICH M7(R1) guidance.

Scheme 10 and Scheme 11 below depict the synthetic Process 2 and Process3, respectively, for X4P-001. Scheme 12 depicts the synthesis of NT-319and AMD-2890 process intermediates.

Table 6 below depicts the identified potentially mutagenic impurities inthe process for manufacturing X4P-001.

TABLE 6 Identified Potentially Mutagenic Impurities in Synthetic Processfor X4P-001.

I-8

I-9

I-10

I-11

The expected duration of treatment for patients in clinical trials isless than 10 years. The acceptable intake of a drug with an exposureof >1 to 10 years is 10 μg/day of a mutagenic impurity, based on Table 2in ICH M7(R1).

Based on a 400 mg/day dose of X4P-001, a limit of up to 25 ppm of apotentially mutagenic impurity is acceptable (10 μg/day/400 mg=25 ppm(μg/g)).

The potential impurities I-10 and I-11 have never been detected inX4P-001. Additionally, a theroretical purge factor calculation onsynthetic process 3, based on published references such as by Teasdaleet al., (Org. Process Res. Dev. 2013, 17, 221), showed that the processefficiently purges both potential impurities I-10 and I-11 with atheoretical purge factor of about 9×10⁸ (I-10) and about 9×10¹⁰ (I-11).

Based on the calculated purge factors, a theoretical residual amount ofI-10 and I-11 of about <0.0001 ppm I-10 and about <0.000001 ppm I-11 inX4P-001 has been calculated, which shows that both potentially mutagenicimpurities are efficiently purged with this process and no control inX4P-001 is required.

The potential impurities RM-396 and AMD-2890 (I-9) have never beendetected (limit of quantitation, or LOQ, 2.5 ppm) in X4P-001, whichshows that both potentially mutagenic impurities are efficiently purgedwith this process.

Example 11: 25 mg, 100 mg, and 200 mg Solid Formulations

During formulation development, the excipients were chosen based onscreening studies involving short term compatibility of variousexcipients. Microcrystalline cellulose was chosen as a diluent/fillerfor 25 mg and lower strength capsules. Dibasic calcium phosphatedihydrate diluent/filler was added to the formulation to improveflowability for the 100 mg higher strength formulation. The ratio ofmicrocrystalline cellulose to dibasic calcium phosphate dihydrate waschosen to approximate the bulk density of drug substance to reduce theprobability of segregation during mixing. Sodium stearyl fumurate waschosen as a lubricant. To facilitate the capsule filling on an automatedcapsule filler, the glidant colloidal silicon dioxide was added to theformulation as a flow aid. Croscarmelose sodium was selected as adisintegrant to enable automated capsule filling. Sodium lauryl sulfatewas added to the formulation to reduce sticking of drug substance duringencapsulation and as a wetting agent.

TABLE 7 Composition of Exemplary X4P-001 25 mg Capsules Reference toQuantity Component Standard Function (mg/capsule) % w/w X4P-001 In HouseActive 25.0 14.7 composition Ingredient Microcrystalline NF Diluent132.7 78.1 Cellulose Croscarmellose NF Disintegrant 10.2 6.0 SodiumSodium Stearyl NF Lubricant 1.7 1.0 Fumarate Colloidal Silicon USP FlowAid 0.4 0.2 Dioxide Sum Total 170.0 100.0 Hard Gelatin USP Packaging NANA Capsules, Size 1

TABLE 8 Composition of X4P-001 100 mg Capsules Reference 100 mg toQuantity Component Standard Function (mg/capsule) w/w X4P-001 In HouseActive 100.0 37.6%  composition substance Dibasic Calcium USP/NF Diluent84.3 31.7%  Phosphate Dihydrate Microcrystalline NF/EP Diluent 60.922.9%  Cellulose Croscarmellose NF/EP Disintegrant 16.0 6.0% SodiumSodium Stearyl NF Lubricant 2.7 1.0% Fumarate Sodium Lauryl NF/EPWetting agent 1.3 0.5% Sulfate Colloidal Silicon NF/EP Flow Aid 0.8 0.3%Dioxide Sum 266.0 100%  Hard gelatin USP Encapsulation N/A N/A capsules,Size 1 white/white. Qualitative composition: Gelatin and Titaniumdioxide.

TABLE 9 Composition of X4P-001 200 mg Capsules 200 mg PercentTheoretical Amount Ingredients Per Capsule (%) Per Capsule (mg) X4P-001composition 61.5 200.0 Microcrystalline Cellulose, 12.9 41.93 NF/EP(Avicel PH 101) or equivalent Dibasic Calcium Phosphate 17.8 57.85Dihydrate, USP/NF Croscarmellose Sodium, NF/EP 6.0 19.50 (Ac-Di-Sol)Sodium Lauryl Sulfate, NF/Ph. 0.5 1.625 Eur. Colloidal Silicone Dioxide,0.3 0.9750 NF/Ph. Eur. (Cab-O-Sil M-5P) Sodium Stearyl Fumarate, NF 1.03.250 (Pruv) Total Capsule Fill 100 325.0

The compatibility of excipients has been shown through ongoing long-termstability studies of the drug product where the product meets stabilityrequirements for the specified refrigerated storage conditions for 3lots of 100 mg capsules. Up to 24-month stability results are availablefor X4P-001 100 mg capsule lot 15K227 packaged in 30 count amber glassbottles and sealed in aluminum foil bags at the storage conditions of 5°C.±3° C. and 25° C./60% RH. At the recommended storage condition of 5°C.±3° C., the results show no significant trends in any stability testparameter up to 24-months (assay, impurities, dissolution, moisture, andmicrobial testing).

Up to 9 months of stability results for 100 mg capsules stored at 5°C.±3° C. and packaged in amber glass bottles sealed in aluminum foilbags or Oxy-Guard HDPE bottles showed an increase in the imine impurityover time, but well within specification. No trends from initial valuesin any of the other stability test parameters were observed.

Up to 3-month stability results for 100 mg capsules stored at 5° C.±3°C. and 25° C./60% RH and packaged in Oxy-Guard HDPE bottles showed anincrease in the imine impurity over time, but well within specification.No trends from initial values in any of the other stability testparameters were observed. These capsules were produced with X4P-001 drugsubstance manufactured using a toluene rework procedure.

The continued use of refrigerated cold chain storage is an anticipatedrequirement for X4P-001 drug product. The X4P-001 drug product primarycontainer packaging to be used in phase 3 clinical trials is theOxy-Guard 60 cc HDPE bottle with 33 mm cap with induction seal. Rayoncoil is placed above the capsules in each bottle and one desiccant pack(0.5 g Sorb-It or equivalent) is positioned at the top of each bottlebetween the Rayon coil and cap (30 count).

A detailed summary of the manufacturing process is provided in FIG. 1.

I claim:
 1. An X4P-001 composition comprising a compound of formula I:

or a pharmaceutically acceptable salt thereof; and the followingcompound:

or a pharmaceutically acceptable salt thereof, in an amount from about0.001 to about 0.5% w/w of the X4P-001 composition.
 2. The X4P-001composition of claim 1, wherein the amount of I-6, or a pharmaceuticallyacceptable salt thereof, is from about 0.01 to about 0.4% w/w of theX4P-001 composition.
 3. The X4P-001 composition of claim 1, wherein theamount of I-6, or a pharmaceutically acceptable salt thereof, is fromabout 0.01 to about 0.3% w/w of the X4P-001 composition.
 4. The X4P-001composition of claim 1, wherein the amount of I-6 is from 0.01 to 0.2%w/w of the X4P-001 composition.
 5. The X4P-001 composition of claim 2,wherein the composition comprises at least a detectable amount of thefollowing compound:

or a pharmaceutically acceptable salt thereof.
 6. The X4P-001composition of claim 2, wherein the composition comprises at least adetectable amount of the following compound:

or a pharmaceutically acceptable salt thereof.
 7. The X4P-001composition of claim 6, wherein the composition comprises at least adetectable amount of the following compound:

or a pharmaceutically acceptable salt thereof.
 8. The X4P-001composition of claim 6, wherein the composition comprises at least adetectable amount of the following compound:

or a pharmaceutically acceptable salt thereof.
 9. The X4P-001composition of claim 8, wherein the X4P-001 composition does notcomprise I-2 or I-5, or a pharmaceutically acceptable salt thereof, in adetectable amount.
 10. The X4P-001 composition of claim 5, wherein theamount of I-2, or a pharmaceutically acceptable salt thereof, is fromabout 0.01 to about 0.3% w/w of the X4P-001 composition.
 11. The X4P-001composition of claim 6, wherein the amount of I-3, or a pharmaceuticallyacceptable salt thereof, is from about 0.01 to about 0.4% w/w of theX4P-001 composition.
 12. The X4P-001 composition of claim 8, wherein theamount of I-3, or a pharmaceutically acceptable salt thereof, is fromabout 0.01 to about 0.4% w/w of the X4P-001 composition.
 13. The X4P-001composition of claim 7, wherein the amount of I-5, or a pharmaceuticallyacceptable salt thereof, is from about 0.01 to about 0.4% w/w of theX4P-001 composition.
 14. The X4P-001 composition of claim 12, whereinthe amount of I-7, or a pharmaceutically acceptable salt thereof, isfrom about 0.01 to about 0.25% w/w of the X4P-001 composition.
 15. TheX4P-001 composition of claim 14, wherein the compound of formula I ispresent in the X4P-001 composition as a free base.
 16. The X4P-001composition of claim 15, wherein the composition comprises no more than3.0% of total impurities.
 17. The X4P-001 composition of claim 16,wherein the R-enantiomeric excess (% ee) of the compound of formula I isno less than 97.0%.
 18. The X4P-001 composition of claim 16, wherein thetotal weight of I-6 and any additional impurities that are presentcomprise no more than about 0.8% w/w of the X4P-001 composition.
 19. Apharmaceutical composition comprising the X4P-001 composition accordingto claim 1, and a pharmaceutically acceptable adjuvant, carrier, orvehicle.
 20. A pharmaceutical composition comprising the X4P-001composition according to claim 15, and a pharmaceutically acceptableadjuvant, carrier, or vehicle.
 21. A compound of the following formula:

or a pharmaceutically acceptable salt thereof.